MOL 345: Final Study Guide

Chapter 27: Integration and Cytoplasm: glycolysis, FA synthesis, and cholesterol synthesis
regulation, Pathways that take place in Inner mito membrane: oxidative phosphorylation
the cytoplasm, IMM, and mitochondrial Mitochondrial matrix: CAC, FA oxidation, ketone bodies synthesis
matrix, interplay between 2 Matrix and cytoplasm: gluconeogenesis; urea cycle
Glucose transporters Work by facilitated diffusion
Glut 1 + 3: in all cells, take up glucose, small Km for constant glucose transport
Work by ____ Glut 2: liver and pancreatic β cells, high Km – sense glucose level and increase
secretion of insulin
Glut 4: transport glucose  muscle and fat (↑ insulin causes ↑ Glut 4 transporters in
plasma membrane, ↑ exercise and ↑ Glut 4 in muscle membrane)
Glut 5: small intestine, fructose transporter
Acetyl CoA A hub for all metabolic pathways
Hub for ___ metabolic pathways Definition: An activated 2-carbon unit
What is it? Derived from: pyruvate (oxidative decarboxylation) and ultimately glucose
Derived from? fatty acids (β -oxidation), catabolism of ketogenic amino acids (those that give rise
Fates? to ketone bodies or fatty acids: Ile, Leu, Trp, Phe, Tyr, Lys)
Fates: Complete oxidation to CO2
Conversion to HMG CoA for synthesis of cholesterol and ketone bodies (in liver)
Conversion to citrate for export from mitochondria to cytosol for synthesis of FAs
Pyruvate A major junction for carbohydrate and amino acid metabolism
A junction for ___ and ___ metabolism Derived from: G6p (glycolysis)
Derived from? lactate (fermentation)
Fates? catabolism of amino acids Gly, Ala, Ser, Cys, Thr, and Trp
Fates: reduction to lactate under anaerobic conditions (to regenerate NAD+ and
allow glycolysis to continue)
transamination to alanine (a major link b/t carbohydrate and aa metabolism)
carboxylation to OAA (in mitochondria) for gluconeogenesis and replenishment of
CAC intermediates
oxidative decarboxylation to acetyl CoA (in mitochondria) for oxidation via CAC,
synthesis of fatty acids
Glucose 6-phosphate A major hub of carbohydrate metabolism
Hub of ___ metabolic pathways Derived from: phosphorylation of glucose, gluconeogenesis (from pyruvate and
Derived from? gluconeogenic aa), glycogen breakdown
Fates? Fates: oxidation to pyruvate when ATP or carbon skeletons are required
Entry into pentose phosphate pathway to produce NADPH for reductive
biosynthesis, Synthesis of glycogen, Dephosphorylation and export from cell (liver)
Brain Neither stores nor produces fuel molecules
Store/production of fuel molecules? In resting state, uses 60% of body’s glucose
Uses what as fuel in resting state? During starvation, uses ketone bodies (fatty aid bound to plasma proteins, cannot
Starvation? cross the blood-brain barrier)
Glucose transporter Glucose transported into brain by GLUT3 (low KM) 0 usually saturated.
Skeletal Muscle Uses glucose during activity, fatty acids while resting
Use ___ as fuel during activity, ___ 1. Synthesizes and stores ¾ body’s glycogen, mobilized during activity
during rest G6P produced by glycogen breakdown, cannot be exported because muscle
Does it synthesize/store fuels? When does not have glucose-6-phosphatase, is charged
does it produce lactate? Where is 2. Production of lactate from pyruvate during active contraction and rate of
lactate transported to? glycolysis >> rate of CAC, NAD+ must be regenerated from NADH for glycolysis to
Where does alanine come into play? continue, Pyruvate + NADH + H+ ↔ lactate + NAD+
Lactate: transported to the liver (Cori cycle): shift metabolic burden to liver
Alanine: Muscle absorbs and transaminates branched aa to use C-skeleton as fuel,
can’t form urea. Nitrogen released into blood as alanine, which the liver converts
into urea and glucose
Adipose Tissue
A major _____ (Why?)
Specialized for ___
Esterification requires ____
__ dictates whether FAs are ___ or ___
When released, FAs and glycerol are _
Function as an endocrine organ?
Synthesis and turnover of
triacylglycerols by adipose tissue
Role of glucose in tissue
Kidney
Uses fuel to? Reabsorption of glucose
via ___, powered by ___
During Starvation, a site of ___
Liver
What passes through liver? Major site
of? Uses ___ as fuel.
Other metabolic functions provided by
liver
Role in regulating lipid metabolism
Why can’t it use branched chain amino
acids, acetoacetate as fuel?
Control sites of Glycolysis
Control sites of Pyruvate
Dehydrogenase Complex
Control sites of CAC
A major storage depot (TAGs stored in adipose tissue – enormous fuel reservoir)
Specialized for esterification of fatty acids and their release from TAGs
Esterification requires glycerol-3-phosphate (glycolytic intermediate)
Intracellular [glucose] dictates whether fatty acids are re-esterified or released
When released, FAs and glycerol are exported to the liver
Endocrine organ: adipose synthesizes peptide hormone leptin, signals fed state in
hypothalamus
Glucose (from liver)  GAP
VLDL (from liver) and Chylomicron (from intestine  fatty acyl CoA
GAP + Fatty Acyl CoA  TAGs
TAGs are hydrolyzed to FAs and glycerol by lipases. (lipase is reversibly
phosphorylated)
Glucose determines whether FA are released in blood (no GAP) or reesterified
Uses fuel to power reabsorption of glucose and water during filtration of blood
plasma (waste products excreted in urine)
Reabsorption of glucose is via glucose symporter, powered by NA+K+ ATPase
During starvation, a site of gluconeogenesis
Most metabolites absorbed by the intestine pass first through the liver
Major site of amino acid deamination and synthesis of urea
Uses α-keto acids (from amino acids degradation) as fuel to yield acetyl CoA
Lactate, alanine (muscle), glycerol (adipose), glucogenic amino acids converted to
G6P, produces glycogen + acetyl CoA (which produces FAs, cholesterol, bile salts)
Fumarate produced by urea cycle yields the OAA necessary for acetyl CoA to be
processed in TCA cycle
When fuels are abundant, FAs are esterified and secreted into blood as low density
lipoproteins. (When Malonyl CoA is abundant, FA can’t enter mito matrix for β
oxidation, instead are exported to adipose tissue to be incorporated into TAGs)
Can’t use branched chain amino acids as fuel because it cannot complete
transamination of amino acid as can muscles.
Can’t use acetoacetate as fuel because it doesn’t has transferase needed to active
acetoacetate to acetyl CoA (used in brain)
PFK (committed site) F6P  F-1,6-BP: (-) ATP, Citrate, H (+) AMP, F26BP
*in liver: (+) F-2,6-BP (produced by PFK2 when blood glucose is high), allosteric
activator of PFK by ↑ velocity at low [substrate] and ↓ responsiveness of
PFK1 to allosteric inhibitor ATP.
Hexokinase (-) G6P
PK (controls outflow of glycolysis)
(+) F1,6BP, (-) ATP, Alanine, phosphorylation by glucagon-stimulated cAMP
cascade (in liver)
Pyruvate  acetyl CoA (pyruvate dehydrogenase) = irreversible link between
carbon atoms of carbohydrates/amino acids to oxidation by CAC/synthesis of lipids
(-) acetyl CoA inhibits transacetylase (E2), NADH inhibits E3 – energy needs of cell
are met or FA are being degraded, save glucose, ATP, phosphorylation by kinase
(+) pyruvate, dephosphorylation by phosphatase (simulated by Ca2+)
Hormones: epinephrine  α-adrenergic receptor  ↑ Ca2+  activates phosphatase
 ↑ PDH
Insulin  ↑ phosphatase  ↑ pyruvate  acetyl CoA  ↑ FA in tissues with FA
synthesis (liver, adipose)
1. isocitrate  α-ketoglutarate (isocitrate dehydrogenase)
(-) ATP, NADH (cooperative inhibition of NAD+)
(+) ADP, NAD+, Mg2+
2. α-ketoglutarate  succinyl CoA (α-ketoglutarate dehydrogenase)
(-) ATP, succinyl CoA, and NADH
3. Acetyl CoA + OAA  Citrate (citrate synthase)

Control sites of oxidative
Phosphorylation
Control sites of gluconeogenesis
Control sites of glycogen synthesis
and degradation
Control sites of FA synthesis
Control sites of FA Degradation
Well Fed state reactions
(-) ATP, ↑ Km for acetyl CoA
Complete oxidation of an acetyl unit by CAC = 1 GTP, 3 NADH, 1 FADH2
Respiratory control – tight coupling of electron transport and ADP
phosphorylation, ensures rate of CAC matches need for ATP
(+) ADP, NADH, FADH2, O2, Pi
F-1,6 BP  F6P (F16BPase)
(-) AMP, F2,6BP
(+) Citrate
Breakdown: Glycogen + orthophosphate  G1P (glycogen phosphorylase)
Synthesis: UTP + G1P  UDP-glucose (UDP-glucose pyrophosphorylase)
1. Protein Kinase A (phosphorylation)
↑ phosphorylase kinase  ↑ glycogen phosphorylase  [↑ glycogen breakdown]
↓ glycogen synthase (ab)  [↓ glycogen synthesis]
2. Protein Phosphatase 1 (dephosphorylation)
↓ phosphorylase kinase  ↓ phosphorylase a  [↓ glycogen breakdown]
↑ glycogen synthase (ba)  [↑ glycogen synthesis]
PKA regulates PP1:
Epinephrine/glucagon  activates PKA:
a. phosphorylate RG1, releases catalytic unit (assoc. needed for PP1 activity)
b. phosphorylated inhibitor binds to catalytic unit of PP1
3. Insulin (activates glycogen synthase kinase, PP1)
Insulin  activates tyrosine kinase receptor in plasma membrane  phosphorylates
IRS  signal cascade  glycogen synthase kinase inactive  active
dephosphorylated glycogen synthase (PP1 also dephosphorylates glycogen synthase)
Insulin phosphorylates RG1 subunit on PP1 (associates with glycogen)
Acetyl CoA + ATP + HCO3-  malonyl CoA + ADP + Pi + H+ (Acetyl CoA
carboxylase) = committed step
- global regulation: hormones change phosphorylation
Active dephosphorylated carboxylase  inactive phosphorylated carboxylase
(AMP-activated protein kinase AMPK)
(+) Glucagon, epinephrine
Inactive phosphorylated carboxylase  active dephosphorylated carboxylase
(protein phosphatase 2A)
(+) insulin (stimulate accumulation of TAGs by muscle and adipose tissue)
- local regulation:
(+) Citrate  polymerizes inactive carboxylase dimers into active filaments,
counteracts phosphorylation (high ATP/acetyl CoA = raw material sand
energy available for FA synthesis)
(-) AMP  turn on AMPK  phosphorylates and inactivates carboxylase (low
energy charge, fats only synthesized when energy is abundant)
(-) Palmitoyl CoA (abundant when FA abundant)  carboxylase filaments
disassemble, inhibits translocase that transports citrate from mit cyto,
inhibits G6Phosphate dehydrogenase, generates NADPH
Malonyl CoA (from carboxylase) --] Carnitine acyltransferase
(block entry of FA CoAs into mito for degradation when synthesizing FA)
NADH --] HMG-CoA dehydrogenase
Acetyl CoA --] thiolase (β -oxidation enzyme inhibited when energy charge is high)
- glucose and aa transported from intestine to blood
- dietary lipids are packaged into chylomicrons and transported to the blood by
lymphatic system, secretion of insulin by β cells in pancreas (stimulated by glucose
and PNS)
- Insulin initiates protein kinase cascades, ↑ glycogen synthesis in muscle and liver,
↓ gluconeogenesis by liver, ↑ glycolysis and synthesis of FAs
 - Liver has glucokinase (high Km, only active when blood-glucose levels are high),
liver forms glucose-6-phosphate more rapidly as blood-glucose levels rises. Increase
in G6P + insulin = ↑ glycogen.
-Phosphorylase a = glucose sensor + cleaves glycogen
- high glucose  ↑ phosphatase activity  inactivates glycogen phosphorylase (a
b): allosteric shift from glycogen degradation to synthesis mode
- high insulin = entry of glucose into muscle and adipose tissue. (glucose  GAP for
TAGs), Insulin promotes uptake of branched-chain aa (val, leu, ile) by muscle,
stimulates protein synthesis, inhibits intracellular protein degradation
Early Fasting Low insulin, high glucagon
Hormones? Processes: Mobilization of TAGs in adipose tissue, gluconeogenesis in liver
Dominant metabolic processes? Changes: liver oxidizes FAs from adipose, muscle shifts to use fatty acids because
Changes in fuel Use? low [insulin] leads to reduced uptake of glucose, the acetyl CoA product inhibits
conversion of pyruvate to acetyl CoA.
Pyruvate, alanine, and lactate are exported to liver for gluconeogenesis
Prolonged Starvation After several weeks, synthesis of large quantities of ketone bodies in liver
After several weeks, synthesis of ___ Acetyl CoA from β -oxidation of fatty acids in liver cannot enter CAC because
Why can’t Acetyl CoA enter CAC? gluconeogenesis limits [OAA], ketone bodies are made and exported
Production of ___ reduces need for __, Production of ketone bodies decreases the need for glucose, less protein is degraded
less ___ is degraded. Death results than during the initial period of starvation
from? Protein degradation accelerates after reserves exhausted, death from loss of heart,
liver, or kidney function
Where does Acetoacetate come from? Acetoacetate is activated by transfer of CoA from succinyl CoA to give acetoacetyl
CoA. Thiolase cleavage yields to molecules of acetyl CoA, which then enters the
Effects of excess Acetyl CoA CAC. Ketone bodies = fatty acids that are an accessible fuel source of the brain.
Glucose  Pyruvate  OAA or Acetyl CoA
Acetyl CoA blocks PDH from making more acetyl CoA, stimulates gluconeogenesis
and production of OAA from pyruvate (pyruvate carboxylase).
Obesity: What is it? Symptoms? Metabolic disorder, BMI > 30, may develop hypertension, cardiovascular disease,
2 neural signals that regulate appetite? type II diabetes
In _____ In hypothalamus a. hormones (leptin – produced from fat in adipose, signals well-
fed state), 2. Ghrelin, peptide YY (made in intestinal cell), released upon eating and
signals hunger and satiation
b. metabolites: 1. glucose (signal for insulin release, low concentration promotes
hunger), 2. free fatty acids and branched aa’s – signal to suppress feeding
Diabetes Type I : Cause: Insulin-dependent
___ dependent auto-immune destruction of β cells in islets of Langerhans in pancreas (early in
Cause life)
Symptom Symptoms: no insulin produced, glucagon overproduced  high glucose,
Treatment permanent “starvation” state
inhibits glycolysis, increases gluconeogenesis in liver, because of PFK2 and
F26BPase = glycogen breakdown
Glucose released into blood, concentration already high
Excreted by kidney = water loss and dehydration
Uncontrolled lipid and protein breakdown
- ↑ acetyl CoA high (lipid)  ketone bodies
- ↑ ketone bodies exceeds acid-base balance in kidney  ↓ pH 
dehydration and coma
1. Without insulin, liver can’t take up glucose, OAA, CAC + β -ox of fatty acids ↓
2. Free FA are released from adipose tissue into bloodstream
4. Liver picks up FA, converts into ketone bodies
5. Ketone bodies are moderately strong acids, blood pH drops
6. Acidosis, Coma + Death
Treatment: administer insulin, control diet and exercise
Diabetes Type II :
Cause
Symptom
Treatment
What controls development of Type II
diabetes?
Compare allosteric interactions and
covalent modifications
CAC provides what intermediates for
biosynthesis?
Reciprocal regulation of
gluconeogenesis and glycolysis
How is compartmentation involved in
FA synthesis/degradation
Chapter 15: Principles of
Metabolism
Define metabolism, catabolism,
anabolism
Three major purposes of living
organisms that require constant input of
free energy
Significance of free-energy change of
reactions and relation to the
equilibrium constant, concentrations of
reactants and products of reaction
Structure of ATP
Role
How can coupling a reaction with the
hydrolysis of ATP change the
equilibrium concentration of the
products to the concentrations of the
reactants by a factor of ___
Compounds that have high group-
transfer potential (define)
Creatine phosphate serves as ___
Cause: Although insulin is made normally, cells can’t respond to hormone (messed
up insulin receptor/production)
Symptom: patients are hyperglycemic (excess blood glucose) because cells do not
take up and utilize glucose normally
Treatment: drugs that stimulate insulin production or responsiveness to hormone
(metfomin), diet and exercise to use up glucose
WT adipocyte (releases Leptin – signals fed state, adiponectin – promotes insulin
action, removes glucose from blood) converted to ‘Fat’ adipocyte
*excess carbohydrates, proteins, or fat converted to FA, stored as TAGs
‘Fat’ adipocyte, stores more TAG, increase number and size (releases retinol
binding protein 4 RPB4, block insulin axn, contributes to insulin resistance, TNF α,
IL6 – inflammatory cytokines, induce chronic, low level inflammation in adipose,
linked to development of insulin resistance)
Allosteric interactions – enable enzymes to rapidly detect diverse signals and adjust
activity accordingly
Covalent modifications – a final step in an amplifying cascade that allows
metabolic pathways to be rapidly switched on or off by low concentrations of
triggering signals. Insulin, glucagon, and epinephrine stimulate protein kinases.
Covalent modifications (s-min) last longer than reversible allosteric interactions (ms
– sec)
Succinyl CoA forms porphyrins and citrate forms fatty acids.
AMP inhibits F16BPase (gluconeogenesis), activates PFK (glycolysis)
Citrate activates F16BPase (gluconeogenesis), inhibits PFK (glycolysis)
F26BP inhibits F16BPase (gluconeogenesis), activates PFK (glycolysis)
Fatty acids are transported into mitochondria for degradation only when energy is
required, whereas fatty acids in cytoplasm are esterified or exported.
Catabolism + Anabolism
108
Compounds that release large amounts of free energy on hydrolysis or oxidation
“high-energy” buffer in vertebrate muscle
ATP-ADP cycle of energy exchange in
biological systems
How does oxidation of carbon
compounds drive the formation of
ATP?
Define proton gradient
How does proton gradient couple
unfavorable reactions to favorable
ones?
Major stages in extraction of energy
from foodstuffs
Why are most high-energy compound
and reduced electron carriers
kinetically stable, despite high-energy
status?
Require __ for reactions enzymes
Structure of CoA, role as ___ Carrier of acetyl/acyl groups

List major activated carriers (electrons

and activated groups) in metabolic
reactions
Six basic types of chemical reactions
encountered in cellular metabolism
Three major mechanisms for the
regulation of metabolism
Energy charge vs. phosphorylation
potential
Common structural features of NAD,
FAD, and ATP
Chapter 16: Glycolysis
Define glycolysis
Three stages of glycolysis
Why phosphorylate glucose?
Hexokinase mechanism upon glucose
binding, two important consequences
of this step in glycolysis
The net reactions for the transformation
of glucose into pyruvate and enumerate
the ATP and NADH molecules formed
Glycolysis = 1 glucose  2 pyruvate + 2 ATP
Stage 1: energy input, trap glucose in cell to be cleaved into 3 C units
Stage 2: cleave F1,6BP into 3C units
Stage 3: ATP output, oxidation to pyruvate
1. G6P can’t pass through membrane, not a substrate for transporters
2. destabilizes glucose, facilitate further metabolism
Induced-fit rearrangement
1. environment around glucose – nonpolar, favors phosphorylation
2. keep out water
1. Glucose + ATP  G6P + ADP + H+ (hexokinase) phosphoryl transfer
2. G6P ↔ F6P (phosphoglucose isomerase) isomerization
3. F6P + ATP  F1,6BP + ADP + H+ (PFK) phosphoryl transfer
4. F1,6BP  GAP + DHAP (aldolase) aldol cleavage
5. DHAP ↔ GAP (TIM) isomerization
6. GAP + NAD+ + ATP  1,3 BPG + NADH + H+ (GAP DH) ox. phosphorylation
7. 1,3 BPG + ADP  3 PG + ATP (Phosphoglycerate kinase), sub level phosphor.
8. 3PG  2 PG (phosphoglycerate mutase), phosphoryl shift
9. 2PG  PEP + H2O (enolase) dehydration
10 PEP  Pyruvate + ATP (pyruvate kinase), substrate level phosphorylation
Rxns for the conversion of pyruvate
into ethanol, lactate, or acetyl CoA.
Role of alcoholic fermentation and
lactate formation
Structure of the NAD+-binding region
common in dehydrogenases
Why does PFK use AMP and not ADP
as positive regulator?
Regulation of glycolysis in skeletal
muscle cells to that in liver cells
Physiologic significance of
gluconeogenesis. Primary precursors of
gluconeogenesis
How do Cancer and Exercise training
affect Glycolysis in a similar fashion?
Different steps in gluconeogenesis.
Enzymes, intermediates, and cofactors
involved in reactions
Major organs that carry out
gluconeogenesis. Location of enzymes
in cell compartments.
Transfer of carboxylase reaction
products
Pyruvate carboxylase: Role of biotin,
Effect of acetyl CoA, maintaining the
level of CAC intermediates
Calculate the number of high-energy
phosphate bonds consumed during
gluconeogenesis, number formed
during glycolysis
Coordinated control of the enzymes in
glycolysis and gluconeogenesis
Fused-domain structure of PFK2 that
forms and degrades F26BP.
Reciprocal regulation of enzyme
Substrate cycle can
1. Ethanol: pyruvate  acetaldehyde + CO2 (pyruvate decarboxylase)
Acetyladehyde + NADH ↔ ethanol + NAD+ (alcohol dehydrogenase)
2. Lactate: pyruvate + NADH ↔ lactate + NAD+ (lactate dehydrogenase)
No oxidation-reduction, regenerate NAD+ for glycolysis to continue anaerobically
Rossman fold
Some ATP can be salvaged from ADP, AMP is signal for low-energy state, provides
an especially sensitive control
Liver PFK: (+) F-2,6BP, (produced when abundant F6P) feed forward stimulation
Glucokinase provides G6P for glycogen + FAs synthesis (lower affinity for glucose)
L and M isozymes of P, L form controlled by reversible phosphorylation:
When BG low, glucagon-triggered cAMP cascade leads to phosphorylation,
decrease activity.
Maintain blood glucose levels
AA (OAA, pyruvate), lactate (pyruvate), glycerol (DHAP)
Glycerol  Glycerol phosphate (glycerol kinase)  DHAP (glycerol phosphate DH)
Cancer cells grow blood vessels, use lactic acid as primary source of ATP, increase
expression of GLUT1 + 3 to uptake glucose.
Anaerobic exercise: enhance blood-vessel growth and generate ATP anerobically
Glycolysis Gluconeogenesis
Glucose + ATP  G6p + ADP G6P + H2O  Glucose + Pi
(hexokinase) (glucose 6 phosphatase)
F6P + ATP  F16BP + ADP (PFK) F16BP + H2O  F6P + Pi (F 1,6
biphosphatase) – hydrolysis
PEP + ADP  Pyruvate + ADP Pyruvate + CO2 + ATP + H2O  OAA
(pyruvate kinase) + ADP + Pi + 2H+ (pyruvate
carboxylase)
OAA + GTP  PEP + GDP + CO2
(PEP carboxykinase)
Liver, kidney
Matrix (PEP carboxykinase, F16Biphosphatase, G6phosphatase) and cytoplasm
(pyruvate carboxylase).
OAA (product of carboxylase reaction) transported to cytoplasm in the form of
malate, which is reoxidized to OAA in cytoplasm.
Biotin = covalently attached biotin group, carrier of activated CO2 in the pyruvate
carboxylase. Biotin is not carboxylated unless acetyl CoA is bound to enzyme.
Six triphosphate molecules are hydrolyzed to synthesize glucose  pyruvate in
gluconeogenesis (G6P, 2 Pyruvate + 4 ATP)
2 molecules of ATP are generated in glycolysis  pyruvate
Extra cost of gluconeogenesis = 4
* energy needed = glycolysis, energy surplus =gluconeogenesis
1. F6P  F1,6BP
Gluconeogenesis: (-) AMP, F-2,6-BP (+) Citrate
Glycolysis: (+) AMP, F-2,6-BP (-) ATP, Citrate, H
2. PEP ↔ Pyruvate
Gluconeogenesis: (-) ADP (+) Acetyl CoA (pyruvate  OAA)
Glycolysis: (+)F-2,6-BP (-) ATP, Alanine
Low BG: F2,6BP loses a phosphoryl group  f6p, doesn’t bind PFK
Phosphorylates bifunctional enzyme – lower level of F26BP, ↓ glycolysis
High BG: F6P accelerates formation of F-2,6-BP , facilitate the dephosphorylation
of bifunctional enzyme

Outline Cori cycle.
Biological significance
Chapter 17: The Citric Acid Cycle
Role of CAC in aerobic metabolism
Pyruvate dehydrogenase as a multi-
enzyme complex
Cofactors that participate in pyruvate
dehydrogenase complex reactions
Enzymatic mechanism of citrate
synthase
Role of iron in the enzyme aconitase
Compare the rxn catalyzed by the α-
ketoglutarate DH to the rxn catalyzed
by pyruvate dehydrogenase complex
Enzymatic reactions of the CAC in
sequence. Name enzymes
-or- Examples of condensation,
dehydration, hydration,
decarboxylation, oxidation, and
substrate-level phosphorylation
reactions
Yield of ATP form the complete
oxidation of pyruvate or of acetyl CoA
Summarize the regulation of the
pyruvate dehydrogenase complex
through reversible phosphorylation
Major activators and inhibitors of the
kinase and phosphatase
Control points of the CAC, activators
and inhibitors
Build up?
CAC intermediates that can be used as
biosynthetic precursors
Roles of anaplerotic reaction
Pyruvate carboxylase reaction
Amplify metabolic signals
Produce heat
Muscles produce lactate, move to liver, gluconeogenesis to produce glucose
transported back to muscles.
Relieve metabolic burden of muscles
Common pathway for oxidation of carbohydrates, fatty acids, and amino acids. Most
enter as Acetyl CoA
Produces CO2 and NADH
E1: TPP prosthetic group –decarboxylation and oxidation of pyruvate
E2: Lipoamide – transfer of acetyl group to CoA
E3: FAD – regeneration of the oxidized form of lipoamide
(catalytic) TPP, lipoic acid, FAD, (stoichiometric) CoA and NAD+
1. decarboxylation of pyruvate by E1-TPP
2. transfer of acetate to lipoamide
3. transfer of acetyl group to CoA
4. regenerate lipoamide and extract electrons
Condensation reaction, binds OAA first, then acetyl CoA, forms an enol
intermediate  citryl CoA complex. Enzyme only cleaves citryl CoA, not acetyl
CoA. *induced fit* prevents undesirable side reactions
Citrate  aconitate  isocitrate (dehydration, hydration), Iron binds to citrate
through COO- group and Oh group. Helps dehydrate, rehydrate substrate
- substrate: Pyruvate, α-ketoglutarate decarboxylation of α-ketoacid)
- formation of a thioester linkage with CoA (high transfer potential
- product: acetyl CoA, succinyl CoA
1. acetyl CoA + OAA + H2O  citrate + CoA + H+ (citrate synthase), condensation
2. citrate ↔ isocitrate (aconitase) hydration, dehydration
3. isocitrate + NAD+  α-ketoglutarate + CO2 + NADH (isocitrate DH), ox. decarb.
4. α-ketoglutarate + NAD+ + CoA  succinyl CoA + CO2 + NADH (α-keto DH),
oxidative decarboxylation
5. succinyl CoA + ADP  succinate + CoA + ATP (succinyl CoA synthase),
substrate level phosphorylation
6. succinate + E-FAD  fumarate + E-FADH2 (succinase dehydrogenase), oxid.
7. fumarate + H2O  malate (fumerase), hydration
8. malate + NAD+  OAA + NADH (malate dehydrogenase), ox.
3 NADH x 2.5 + 1 FADH2 x 1.5 = 9 ATP + 1 ATP = 10 ATP
(-) acetyl CoA (bind E2), NADH (inhibits E3), ATP – energy needs of cell are met,
fatty acids are being degraded, no need for pyruvate to acetyl CoA
Phosphorylation (kinase, associated with E2) of E1 switches off activity
Dephosphorylation (phosphatase) of E1 switches on activity
(+) ADP, pyruvate, Ca2+, epinephrine
Isocitrate dehydrogenase: (+) ADP, (-) ATP, NADH
α –ketoglutarate dehydrogenase: (-) ATP, NADH, succinyl CoA
Inhibited isocitrate DH = buildup of citrate, transported to cytoplasm to stop
glycolysis, source of Acetyl CoA for FAs (ATP-citrate lysase)
OAA: Glucose, aspartate, amino acids, purines, pyrimidines
Citrate: Fatty acids, sterols
α-ketoglutarate: glutamate, other amino acids, purines
Succinyl CoA: porphyrins, hem, chlorophyll
Replenishes pathway components
Pyruvate carboxylase produces OAA from pyruvate for CAC
Consequences and biochemical basis of
thiamine deficiency.
Compare the effects of heavy-metal
poisoning with mercury or arsenite
Chapter 18: Oxidative
Phosphorylation
Define oxidative phosphorylation and
respiration
Energy yield from glycolysis and
CAC?
The coupling of ____ by a ___ (____)
forms ATP
Energy transfers of CAC, oxidative
phosphorylation
Respiratory chain
Compartments and membranes of
mitochondria, respiratory assemblies
and the N and P sides of the inner
membranes
Quantitatively relate redox potential
(∆ E0’) and free energy change (∆ G0’)
Redox potential (∆ E0’) of a redox
couple relative to the standard
reference half-cell
Free energy stored in concentration
gradients
Calculate energy of proton gradient
Components of the respiratory chain
and electron-carrying molecules
Describe the entry of electrons from
NADH into NADH-Q oxidoreductase
(Complex I)
Trace electron path through proton
pump
What groups transfer electrons?
Role of coenzyme Q as a mobile
electron carrier between NADH-Q
oxidoreductase and cytochrome
reductase (Complex III)
Beriberi – no thiamine (vitamin B), precursor of TPP (cofactor of prosthetic group of
pyruvate, α-ketoglutarate DH, transfer activated aldehyde unit), neurons use only
glucose, so enzyme deactivation = no fuel, other tissues can use fats in CAC
Mercury + arsenite have high affinity for neighboring sulfhydryls (those in
dihydrolipoyl groups of E3, inhibits complex = CNS disease.
Oxidative Phosphorylation: process in which ATP is formed as a result of transfer of
electrons from NADH or FADH2 to O2 by a series of electron carriers in IMM
Respiration: the release of energy from glucose inside living cells
4 ATP, and 10 NADH + 2 FADH2
The coupling of oxidation of fuels to phosphorylation of ADP by a protein
gradient (proton-motive force) forms ATP
CAC: produce electrons with high transfer potential
Ox phos: electron-motive force converted to proton-motive force, then to a
phosphoryl-transfer potential.
3 electron-driven proton pumps (NADH-Q oxidoreductase, Q-cytochrome c
oxidoreductase, and cytochrome c oxidase) convert electron-motive force into
proton-motive force. Contain quinones, flavins, iron-sulfur clusters hemes, and Cu2+.
OMM: has mitochondrial porin, permeable to small molecules
IMM: impermeable to ions/polar molecules
Transporter shuttles: ATP, pyruvate, citrate
Matrix side (-), cytoplasmic side 9P)
∆ G0’ = -nF∆ E0’ (n = # electrons, F = 96.48 kJ/mol V)
∆ G0’ unit is kJ or kJ/mol, ∆ E0’ unit is volts
Measure electromotive force generated by a sample half-cell connected to a standard
reference cell. If electrons flow to standard reference, reduction potential is negative.
Reducing agent (NADH) = negative, Oxidizing agent (O2) = positive reduction pot.
∆ G = 2.303RT log10(C2/C/1) + ZF∆ V
C2 = [destination], C1 = [origin], R = 8.315E-3 kJ/mol, Z = charge of species, ∆ V =
membrane potential
∆ G = 2.303RT log10(C2/C/1) + ZF∆ V
If pH is 1.4 units lower outside than inside, log10(C2/C/1) = 1.4 (more H+ outside)
I. NADH-Q oxidoreductase
II. Succinate-Q reductase (contains succinate DH, generates FADH2)
III. Q-cytochrome c oxidoreductase
IV. cytochrome c reductase
I, III, IV: e- flow through transmembrane complex  transport of H+ across IMM
Electrons carried by reduced form of coenzyme Q (ubiquinone, hydrophobic,
diffuses rapidly in IMM, five isoprene unit tail)
[Cytochrome c (small soluble protein) shuttles electrons from Q-cytochrome c
oxidoreductase  cytochrome c reductase (reduces oxygen)]
NADH binds complex I, transfers 2 high potential electrons to FMN prosthetic
groups  Fe-S clusters (undergo oxidation-reduction w/o proton exchange)  Q
*4 H+ pumped out of matrix of mitochondria, Q accepts 2 electrons = QH2, leaves in
hydrophobic interior of the membrane
Quinone can exist in 3 oxidation states
Q = fully oxidized, two ketones
QH· = 1 e- and 1 H+, semiquinone, Q·- = lose hydrogen, semiquinone radical anion
QH2 = 2 e- and 2 H+, ubiquinol (fully reduced)
Q pool: a pool of Q and QH2 in IMM
Describe the entry of electrons into the
respiratory chain at the succinate-Q
reductase complex (Complex II) from
Is Complex II a proton pump?
The prosthetic group and functions of
cytochromes.
Contrast features of cytochromes b, c1,
c, a, and a3
Components of the Q-cytochrome c
oxidoreductase complex.
How does ubiquinol transfer its
electrons to cyt c1 and b and ultimately
to cyt c?
Role of heme?
How does a two-electron carrier
(ubiquinol) interact with a one-electron
carrier (Fe-S cluster)
Describe the cytochrome oxidase
proton pump (Complex IV) and its
electron carrying groups
Outline the mechanism for the
reduction of oxygen to water on
cytochrome oxidase
Describe the path of the electrons
Chemiosmotic model of oxidative
phosphorylation and relate
experimental evidence that only the
proton-motive force links the
respiratory chain ad ATP synthesis.
Mitochondrial location, subunit
structure, and function of eukaryotic
ATP synthase
Mechanism of ATP synthesis by ATP
synthase during proton flow.
Catalytic cooperativity’s relation to
CAC: succinate  fumarate (succinate DH), part of succinate-Q reductase complex
FADH2 electrons transferred to Fe-S centers  Q  ETC
*does not transport protons, less ATP is made from FADH2 than NADH
No.
Cytochrome water-soluble, electron-transferring protein w/ a heme prosthetic grp.
(Iron ion reduced ferrous +2 state  ferric +3 state)
cyt b, c1, cyt c: iron-protoporphyrin IX (like Mg and HB), different electron affinities
(cytoplasmic side = lower affinity for electron than matrix)
3 heme groups = b and c + iron-sulfur protein (Rieske center), stabilizes reduced
center so it can accept electrons from QH2
Cycle a: 1st QH2 molecule binds complex, gives up 2 electrons (one to cyt c1  cyt
c, one to bound Q  Q·-) and releases two protons New Q enters Q pool.
Cycle b: 2nd QH2 gives up electrons (1 to cyt c, another to reduce Q·- to QH2). And
takes up two protons from matrix.
1 cycle = 2 QH2 molecules oxidized, 1 Q molecule reduced to funnel electrons from
two=electron carrier to 1 electron carrier (cyt c). Cyt b = a recycling device in
reductase that enables both electrons of QH2 to be used effectively.
QH2 electron flows to Rieske 2 Fe-2S cluster  cyt c1  cyt c.
Transports electrons from cyt c to molecular oxygen
4 electrons funneled to O2 to produce H2O, 4 protons pumped to cytoplasmic side of
IMM, ∆ G°’ = -231.8 kJ, energy captured as proton gradient, used in ATP synthesis
Cyc c oxidase has 2 heme A groups, 3 copper ions: CuA/CuA (accepts electrons from
reduced cyt c), CuB coordinated by 3 histidine residues (alternates between cuprous
Cu+ and Cu2+ cupric form when accepting electrons.)
**Heme a3 (Fe) and CuB is active center where O2 is reduced to H2O.
Heme A in cyt c oxidase different from cyt c because heme is not covalently
attached to the protein.
1. Electrons from 2 cyt c  CuA/CuA  Heme a  CuB and heme a3.
2. O2 binds and abstracts an electron from each nearby ion = peroxide bridge.
3. Two more cyt c bind and release electrons that travel to active center. Two
protons cleave the peroxide bridge.
4. Addition of two more protons leads to release of water.
Chemiosmotic hypothesis: electron transport and ATP synthesis are coupled by a
proton gradient across the IMM, protons flow back into matrix and equalize
concentration gradient. Proton motive force = pH and charge gradient. pH is 1.4
units lower inside, membrane potential is .14 V (outside positive).
Test: Synthetic vesicle with bacteriorhodopsin (light-driven proton pump) +
mitochondrial ATP synthase. Exposed to light, ATP was formed, showed respiratory
chain and ATP synthase are biochemically separate systems linked only by a proton-
motive force.
ATP synthase = Complex V.
Fo subunit: in IMM “stick”: proton channels of complex, 10-14 c subunits
embedded in membrane. A subunit binds outside of the ring.
F1 subunit: “ball” = catalytic ATPase activity: α3β 3, central stalk: ε + γ (breaks
symmetry), extends into α3β 3 hexamer, δ
2 ways Fo and F1 are connected: by central γ ε stalk + exterior column
Exterior column = a subunit, 2 b subunits and δ subunit (prevent hexamer
rotation)
Moving = c ring + γ ε stalk, stationary: hexamer
ATP forms in absence of proton-motive force, but doesn’t leave catalytic site unless
proton flows through enzyme. Proton gradient helps release ATP from synthase.
Binding-change mechanism: (Boyer) β subunit performs 3 sequential steps in
binding-change mechanism?
Evidence for rotational catalysis
How does proton flow from Fo drive
the rotation of the γ subunit?
ATP yields for NADH, FADH2
Is IMM permeable to NADH?
Implications?
ATP-ADP translocase mechanism and
energy cost
In an actively respiring mitochondrion,
Net yield of ATP from the complete
oxidation of glucose, different shuttles
for the cytoplasmic reducing
equivalents of NADH
Source of uncertainty in estimation
Respiratory control
Relation to energy charge
ATP synthesis by changing conformation:
1. ADP + Pi binding: loose conformation
2. ATP synthesis: tight conformation, bind ATP so tightly ADP and Pi are converted
3. ATP release: open conformation (LTO)
System with only α3β 3γ subunits, β subunit has amino-terminal polyhistidine tag
(high affinity for nickel ion), immobilized on glass surface coated with nickel. γ-
subunit linked to fluorescently labeled actin filament (long stem that can be observed
under fluorescence microscope). Add ATP causes actin filament to rotate
unidirectionally in a CCW direction. γ subunit rotation driven by hydrolysis of ATP.
Catalytic activity of an individual molecule observed. Lower [ATP] showed γ
subunit rotates in 120 ° increments = hydrolysis of 1 ATP molecule.
a subunit = 2 hydrophilic half-channels that do not span membrane, next to
membrane spanning ring of c subunits
c subunit = a pair of α helices that span the membrane, aspartic residue found in
middle of one of the helices.
1. proton rich environment: proton enters a channel and binds the aspartate residue.
2. Subunit w/ bound proton (neutralized) rotates through membrane, occupies the
hydrophobic environment of the membrane.
3. Proton-poor environment: aspartic acid releases proton.
**c ring is tightly linked to γ and ε subunits. As c ring turns, stalk turns inside
hexamer of F1, promotes binding-change mechanism.
NADH = 2.5 mol of ATP, FADH2 = 1.5 mol of ATP
IMM impermeable to NADH and NAD+. Electrons from NADH enter the matrix via
shuttles (Malate-aspartate shuttle, Glycerol 3-phosphate shuttle)
ATP-ADP translocase = transport protein (antiporter) that couples ATP and ADP
flow. Positive membrane potential increases rate eversion from matrix to cystolic
side when ATP is bound. ¼ of energy yield from electron transfer by respiratory
chain is consumed to regenerate membrane potential tapped by this process.
Glycolysis:
Phosphorylation of glucose -1
Phosphorylation of F6P -1
Dephosphorylation of 2 molecules of 1,3 BPG +2
Dephosphorylation of 2 molecules of PEP +2
Oxidation of 2 molecules of glyceraldehyde 3-phosphate 2 NADH
Pyruvate  Acetyl CoA 2 NADH
CAC:
2 molecules of Succinyl CoA  Succinate +2
Oxidation of 2 molecules of isocitrate, α-ketoglutarate, malate 6 NADH
Oxidation of 2 molecules of succinate 2 FADH2
Oxidative Respiration
2 NADH from glycolysis x1.5 +3
2 NADH from pyruvate  acetyl CoA x 2.5 +5
2 FADH2 from CAC +3
6 NADH from CAC x 2.5 +15
Net ATP yield/molecule of glucose 30 ATP
Stoichiometries of proton pumping, ATP synthesis and transport processes (glycerol
3-phosphate shuttle vs. malate –aspartate shuttle) do not have fixed values.
Electrons only flow through ETC to O2 if ADP is simultaneously phosphorylated to
ATP. ↑ ADP (active muscle), ox phos ↑
Respiratory control: regulation of rate of ox phos by ADP level
Compounds that block electron
transport and locate sites of action
Other uses of proton gradients
Chapter 21: Glycogen Metabolism
Structure of glycogen, location?
Three steps of glycogen catabolism,
fate of its product, glucose-6-phosphate
Precursors of glycogen anabolism
Phosphoglucomutase mechanism
Role of hormones in regulating
glycogen metabolism
Reaction catalyzed by glycogen
phosphorylase
Rxn driven by?
Advantage of Phosphorolytic cleavage
of glycogen over hydrolytic cleavage
Steps in the degradation of glycogen
Importance of glucose 6-phosphatase in
the release of glucose by the liver
Absent in __ and ___.
Why this tissue distribution
Roles of pyridoxal 5’-phosphate, ____
catalysis
Processivity? How does it relate to
glycogen phosphorylase activity
Two primary regulatory mechanisms
for glycogen phosphorylase
Difference in liver and muscle
isozymes of glycogen phosphorylase
Phosphorylase kinase regulation
Role of G-proteins in covalent
modifications
1. Block Electron transport:
Rotenone, amytal block electron transfer in NADH-Q oxidoreductase
Antimycin A – interferes with electron flow from Q-cyt c oxidoreductase  cyt c
CN-, azide, CO can block electron flow from cyt c  O2
2. Inhibit ATP synthase: Oligomycin, DCCD
3. Uncouple electron transport from ATP synthesis: DNP (carry proton across IMM
down concentration gradient, dissipates proton-motive force = heat, no energy)
4. Inhibit ATP export: atractyloside, bongkrekic acid inhibit ATP-ADP translocase
Active transport of Calcium ions by mitochondria, entry of aa and sugars into
bacteria, rotation of bacterial flagella, transfer of electrons from NADP+ to NADPH,
generate heat in hiberantion
Glycogen: branched polymer of glucose, mostly α-1,4 glycosidic linkages and α-1,6
glycosidic linkages (branch point) α-linkages = open helical polymers
β linkages: straight strands (fibrils/cellulose), Liver (10%), skeletal muscle (2%)
1. Glycogen  G1P (glycogen phosphorylase)
2. remodel glycogen to permit further degradation
3. G1P  G6P (phosphoglucomutase)
Glucose (Glucose 6-phosphatase in liver), glycolysis, pentose phosphate pathway
UTP + G1P, UDP-glucose is added to nonreducing ends of glycogen
phosphate on enzyme’s Ser transferred to G1PG1,6,BPG6P(~PG mutase)
Epinephrine, glucagon ↑ breakdown, ↓ synthesis
Insulin ↓ breakdown, ↑ synthesis
Cleaves α-1,4-linkage using orthophosphate  (phosphorolysis), removes glucosyl
residues from nonreducing end (b/t C-1 and C4), stops 4 residues from branch point
Phosphorolysis is favored by high [Pi]:[G1P] ratio > 100
Phosphorolytic cleavage of glycogen gives a phosphorylated sugar that can go down
glycolytic pathway. No transporters exist for G1P in muscle cells, stays in cells.
Transferase shifts block of three glucosyl residues from one outer branch to other.
Α-1,6-glucosidase hydrolyzes the α-1,6-glycosidic bond.
**α-1,6-glucosidase and transferase are on one polypeptide chain.
Liver’s job is to keep a constant level of glucose in blood, glucose 6-phosphatase
enables glucose to leave liver. Absent in brain and muscle because retain glucose for
the generation of ATP (main source of fuel) In contrast, liver uses α-ketoacids as
main source of fuel.
5’ phosphate group of PLP acts in tandem with orthophosphate by serving as a
proton donor and then a proton acceptor (general acid-base catalyst)
Processivity = enzyme that can catalyze many reactions without having to
disassociate and re-associate after each catalytic step.
Glycogen binding site is 30 Å away from catalytic site, connected by a narrow
crevice that accommodates 4-5 glucose units. Enzyme can phosphorolyze many
residues without having to dissociate/re-associate.
Intxns w/ allosteric effectors (signal energy state)/reversible phosphorylation
(response to hormones), isozyme: differ in catalytic properties/response to regulator
Liver enzyme (a/R form) is allosterically inactivated by glucose (T form) –
produces glucose for use by other organs/tissues, conserves glycogen when glucose
is plentiful, a-form is phosphorylated by phosphorylase kinase, hormone: glucagon
Muscle enzyme (b/T form is predominant) ATP, G6P are negative allosteric factors
that stabilizes T state, dephosphorylated by PPI, hormone: epinephrine
Activated by Ca2+ in δ calmodulin and phosphorylation of β subunit by PKA
Hormone 7TM receptor  G protein activates adenylate cyclase ATP to cAMP
 activate PKA  activates phosphorylase kinase  activates phosphorylase a
α –adrenergic receptor initiates phosphoinositide cascade (induces release of Ca2+

Phosphorylation of phosphorylase by
___ and dephosphorylation by ____
Molecular bases for the relative
inactivity of the T state
Steps in the synthesis of glycogen,
pertinent enzymes
Functional importance of branching in
the glycogen molecule
Efficiency of glycogen as a storage
form of glucose
Regulation of glycogen synthase.
Effects of phosphorylation on glycogen
synthase and glycogen phosphorylase
Role of protein phosphatase I in control
of the activities of glycogen
phosphorylase and glycogen synthase.
How is it regulated?
Effects of insulin on glycogen. Why
does a distinct pathway exist for the
biosynthesis and degradation of
glycogen?
After meal, what happens to
phosphorylase a and glycogen synthase
b/c of the presence of glucose in the
liver?
Provide examples of glycogen storage
diseases; relate the biochemical defects
with the clinical observations.
Chapter 22: Fatty Acid Metabolism
Define Fatty acid
Four major physiological functions
they serve
from ER)
phosphorylase kinase
protein phosphatase 1 (PP1)
T state is less active because catalytic site is partly blocked. R state – catalytic site is
more accessible.
1. Priming (Glycogenin = dimer of α-1,4-glucose residues, catalyzes addition of 8
glucose units from UDP-glucose to other subunit in dimer = autoglycosylation,)
2. UDP-glucose + Glycogenn  UDP + Glycogenn+1 (glycogen synthase)
3. branching enzyme moves blocks of 7 residues to interior site, α-1,6 linkages
Increases the solubility of glycogen, creates a large number of terminal residues (site
of action of glycogen phosphorylase and synthase = ↑ rate of synthesis/degradation)
1 ATP hydrolyzed to incorporate G6P into glycogen (activate G1P = synthesis)
10% : 1 ATP used to phosphorylate glucose molecules to G6P. (breakdown)
Complete oxidation of G6P = 31 molecules of ATP
Overall efficiency = 97%
Active Glycogen synthase a: dephosphorylated (active w/ or w/o G6P)
Inactive Glycogen synthase b: phosphorylated (active only with high G6P)
Phosphorylation has the opposite effect on glycogen phosphorylase
PPI dephosphorylates, inactivates phosphorylase a + phosphorylase kinase, ↓
glycogen breakdown + activates glycogen synthase b  a, ↑ glycogen synthesis
Catalytic unit of PPI bound to GM/GL regulatory unit (scaffolds that bring
phosphatase + glycogen). To ↓ PPI activity during glycogen degradation,
Epinephrine/glucagon  cAMP  PKA activated 
1. GM phosphorylated and releases catalytic subunit
2. inhibitor phosphorylated and inactivates PPI catalytic subunit.
To ↑ PPI activity during glycogen synthesis, Insulin  activated Tyr Kinase 
Protein Kinase phosphorylates RG1 of PPI, which associates with glycogen.
Insulin stimulates the synthesis of glycogen
insulin binds receptor tyrosine kinase  phosphorylates IRSs  protein kinases
phosphorylate and inactivate glycogen synthase kinase  glycogen synthase no
longer phosphorylated (also dephosphorylated by PPI = restore glycogen reserves)
Enables both pathways to be thermodynamically favorable at all times. Biosynthetic
pathway is exergonic by coupling to hydrolysis of a sufficient # of ATP molecules.
Phosphorylase a is the glucose sensor in liver cells, when glucose binds, shifts from
R  T form. And the participation of phosphorylase a and PP1 in glucose sensing.
Ab results in release of PPI, free to activate glycogen synthase and
dephosphorylate glycogen phosphorylase. **binding of phosphatase to
phosphorylase a prevents premature activation of glycogen synthase
Pt with hypoglycemia (glycogen storage disease, low blood-glucose between meals),
due to lack of glucose 6-phosphatase from liver, glucose cannot be formed from
G6P. Excess G6P in liver causes glycolysis = high level of lactate and pyruvate in
blood. Pts depend on fat metabolism. –OR – Mutation in glucose 6-phosphate
transporter – transport G6P to lumen of ER to be hydrolyzed by phosphatase
Lack α-1,6-glucosidase can only use outermost branches of glycogen. Only fraction
of glycogen is active as a store of glucose
Lack muscle phosphorylase – limit pt’s ability to perform strenuous exercise, pH
usually drops in muscles because of lactate. McArdle disease = alkaline because of
breakdown of Creatine phosphate. Low glycolytic rate
Fatty acid = long hydrocarbon chain + terminal carboxylate group
1. fuel molecules, stored as TAGs, and mobilized to meet energy needs, insulation
2. Fas are building blocks of phospholipids and glycolipids (biological membranes)
3. Proteins modified by FA are targeted to a membrane location

Structure of a triacylglycerol
How are lipids transported?
Overview of degradation of fatty acids,
list chemical reactions used
Overview of synthesis of fatty acids,
list chemical reactions used
Why are TAGs highly concentrated
forms of stored metabolic energy
Adipocyte
Chylomicron
Bile Salts
Lipases
Role of cAMP in regulation of lipase in
adipose cells
Serum album carries ___
Reactions of Glycerol
Physiological importance of these
reactions
Reaction that links CoA to a fatty acid
Where located? Enzyme?
Involvement of carnitine in transport of
fatty acids from cytoplasm into
mitochondria
Four reactions of the β -oxidation
pathway of fatty acid catabolism,
substrates and products
Function of NAD+ and FAD in
reactions
Different Acyl CoA dehydrogenases
Energy yield in ATP molecules for β
oxidation of palmitoyl CoA
Two reactions used to oxidize naturally
occurring unsaturated fatty acids
4. FA derivatives = hormones and intracellular messengers
Uncharged esters of Fas + glycerol
Free fatty acids and monoacylglycerols are absorbed by intestinal epithelial cells,
resynthesized into TAGs, and packaged into chylomicrons. Chylomicrons are
released into lymph system, bind lipases, degraded into FA and monoacylglycerols
and transported into tissue. TAG resynthesized in cell and stored or oxidized to
provide energy in muscle.
1. FA activated
2. Oxidation to introduce a double bond
3. Double bond is hydrated to introduce a hydroxyl group
4. Alcohol is oxidized to a ketone
5. FA thiolysis by CoA  Acetyl CoA + FAn-2 [OHOT]
1. Condensation of activated acyl group + malonyl unit  4C
2. Caronyl group reduced
3. Dehydration
4. Reduction  butyryl CoA [CRDR]
They are reduced and anhydrous (nonpolar)
Adipocyte: fat cell that store triacylglycerols, hormones
Chylomicrons: carry fatty acids from intestine to other tissue
Bile salts: amphipathic molecules synthesized from cholesterol I the liver and
secreted from gall bladder. Incorporate TAG into micelles
Lipase: intestinal enzymes secreted by pancreas that degrade TAGs to free Fas and
monoacylglycerol.
Lipases activated by glucagon, epinephrine, and adrenocorticotropic hormone via
7TM signal transduction cascade, inhibited by insulin.
Glucagon/epinephrine  7TM receptor in adipose tissue  activate adenylate
cyclase  increase cAMP  stimulate PKA  activate lipase by phosphorylation
Serum album carries fatty acids from adipocytes to other tissues.
Liver absorbs glycerol  glycerol 3-phosphate  DHAP (glycolysis/gluconeogenesis)
Glycerol can be converted into pyruvate or glucose in liver (interconvertible
glycerol and glycolytic intermediates)
FA + ATP ↔ Acyl Adenylate (enzyme bound) + Ppi
Acyl Adenylate + HS-CoA ↔ Acyl CoA + AMP
Location: outer mitochondrial membrane, catalyzed by Acyl CoA synthetase
FA must be oxidized in mitochondrial matrix.
Acyl CoA + Carnitine  Acyl carnitine (carnitine acyltransferase I – bound to
outer mitochondrial membrane)
Acyl carnitine shuttled across inner mitochondrial membrane by translocase
Acyl Carnitine  Carnitine + Acyl CoA (carnitine acyltransferase II )
1. ox: acyl CoA + FAD  trans ∆ ²-Enoyl CoA + FADH2 (acyl CoA DH)
2. hyd: trans ∆ ²-Enoyl CoA + H2O  L-3-hydroxyacyl CoA (enoyl CoA hydratase)
3. ox: L-3-hydroxyacyl CoA + NAD+ 3-ketoacyl CoA + NADH (L-3-H CoA DH)
4. thiolysis: 3-ketoacyl CoA + HSCoA  Acyl CoAn-2 + Acetyl CoA (β -
ketothiolase)
C12 – C18 FA oxidized by long chain Acyl CoA dehydrogenase
C4 – C14 FA oxidized by the medium chain Acyl CoA dehydrogenase
palmitoyl CoA (C16)
108 – 2 (ATP to activate palmitate) = 106 ATP/1 molecule palmitate
*db bond is not a substrate for Acyl CoA dehydrogenase – prevent formation of a
C2-C3 db bond.

Oxidation reactions of odd-numbered
fatty acid
When can acetyl CoA enter CAC? If
not?
Name and structure of ketone bodies,
Where produced? Why available to
other organs?
Describe the synthesis of the ketone
bodies.
The role of ketone bodies in normal
human metabolism
Describe the catabolism of the ketone
bodies.
The effect of high levels of acetoacetate
on fat metabolism in adipose tissue
Biochemical basis for the inability of
animals to convert fatty acids into
glucose
Contrast fatty acid oxidation and fatty
acid synthesis
Substrates and products of the
committed step in fatty acid synthesis
and catalytic mechanism.
Role of biotin in the acetyl CoA
carboxylase reaction
The four reactions of the elongation
cycle of fatty acid synthesis
Round Two of synthesis
End of FA synthesis?
Domains of Animal Fatty Acid
Synthase multifunctional enzyme
1. isomerase
2. reductase
Yields Propionyl CoA in final thiolysis step
Propionyl CoA  Succinyl CoA (Propionyl CoA carboxylase) to enter CAC
Can oxidize Acetyl CoAs in CAC only when balanced with carbohydrate
degradation. Otherwise, form Ketone Bodies.
When fasting, starving, diabetes, OAA  glucose and is unavailable for
condensation with acetyl CoA. Acetyl CoA diverted to form D-3-hydoxybutyrate
and acetoacetate (ketone bodies) Ketone bodies produced in liver. Liver lacks CoA
transferase
2 Acetyl CoA  CoA + Acetoacetyl CoA (thiolase)
Acetoacetyl CoA + H2O + acetyl CoA  HMG-CoA + CoA (HMG synthase)
HMG-CoA  Acetoacetate (HMG CoA cleavage enzyme) + acetyl CoA
Acetoacetate  D-3-Hydroxybutyrate (D-3 DH) + Acetone (decarboxylation)
Ketone bodies = water-soluble, transportable form of acetyl units
Favorite source of fuel for heart muscle + renal cortex, brain during starvation
Acetoacetate + Succinyl CoA  Acetoacetyl CoA + Succinate (CoA Transferase)
Acetoacetyl CoA + CoA  2 Acetyl CoA (Thiolase)
High levels of acetoacetate in blood signify an abundance of acetyl units, leads to a
decrease in the rate of Lipolysis in adipose tissue.
Pyruvate + CoA + NAD+  acetyl CoA + CO2 + NADH + H+ = Irreversible!
2 C’s of acetyl group enter CAC, but 2 C leave CAC in decarboxylations, OAA is
regenerated, not formed de novo when acetyl unit is oxidized by CAC.
Plants have enzymes that can convert acetyl CoA  OAA
FA Synthesis FA Degradation
Cytoplasm Mitochondrial matrix
Intermediates linked to sulfhydryl Covalently attached to sulfhydryl
groups of ACP (acyl carrier protein) groups of CoA
Enzymes joined in 1 ppt chain: fatty Enzymes not associated
acid synthase
Activated donor = malonyl ACP, Activated donor: Acyl CoA
driven by release of CO2
Reductant is NADPH Oxidants are NAD+ and FADH
Elongation stops at palmitate (C16) Degradation stops at Acetyl CoAs
1. Committed step: Acetyl CoA + HCO3 + ATP  Malonyl ACP + ADP + Pi + H+
-
(Acetyl CoA carboxylase – contains biotin covalently attached to ε amino of Lys)
2. Acetyl CoA ↔ Acetyl ACP (Acetyl Transacylase – AT)
3. Malonyl CoA + ACP ↔ malonyl ACP + CoA (Malonyl Transacylase)
4. Condensation: Acetyl ACP + malonyl ACP  acetoacetyl ACP + ACP + CO2
(Acyl-malonyl ACP condensing enzyme)
5. Reduction: Acetoacetyl ACP + NADPH + H+ ↔  D-3-hydroxybutryl ACP +
NADP+ (β -ketoacyl ACP reductase)
6. Dehydration: D-3-hydroxybutryl ACP ↔ Crotonyl ACP + H2O (3-hydroxyacyl
ACP dehydratase)
7. Reduction: Crotonyl ACP + NADPH + H+  butyryl ACP + NADP+ (enoyl ACP
reductase)
Butyryl ACP + malonyl ACP  C6 ACP
 C16 ACP (good substrate for thioesterase, hydrolyzes C16 ACP 
palmitate + ACP)
Domain 1: substrate-entry and condensation unit:
Acetyl transferase, malonyl transferase, β -ketoacyl synthase/condensing enzyme
complex.
Advantages
Compare ACP and CoA
Malonyl CoA provides ___
The energy cost of the synthesis of a
Palmitate
Enzymatic machinery for FA synthesis
in bacteria vs. eukaryotes
Describe the transport of acetyl groups
across the inner mitochondrial
membrane, why not use carnitine?
When is NADPH synthesized?
Sources of NADPH used in fatty acid
synthesis, pathways of FA synthesis
Different modes of regulation of acetyl
CoA carboxylase
Reciprocal control of fatty acid
synthesis and degradation through local
(intracellular), hormonal, and adaptive
regulation
Chapter 23: Amino Acid Metabolism
Fate of exogenously supplied amino
acids that are not use for biosynthesis
Where do amino acids come from?
What are they used for?
Half-lives of proteins in mammals
Define Ubiquitin
Function of E1, E2, E3 that participate
in the attachment of ubiquitin to
proteins targeted for degradation
N-terminal rule
Domain 2: reduction unit: acyl carrier protein, β -ketoacyl reductase, dehydratase,
and enoyl reductase
Domain 3: palmitate release unit: thioesterase
(+) coordinate different reactions, efficient intermediate transfer from sites without
leaving assembly, enzymes more stable when covalently joined
Both have a phosphopanthetheine as reactive units
Malonyl CoA provides the driving force for the condensation of acetyl units with the
growing acyl chain by releasing CO2
8 molecules of Acetyl CoA, 15 molecules of NADPH, and 7 molecules of ATP
Eukaryotic synthases are linked in 1 polypeptide chain, forms a dimer
Fas made in cytoplasm, acetyl CoA made from pyruvate in mitochondria.
To transfer acetyl CoA from to cytoplasm, citrate carries acetyl groups across IMM.
Acetyl CoA + OAA  citrate, in cytoplasm, citrate is cleaved by ATP-citrate lyase
(cleave C-C, C-O, or C-N bonds by elimination, forms double bond), cost 1 ATP
Carnitine only carries long-chain FA in degradation.
1. OAA +NADH + H+ ↔ malate + NAD+
2. Malate + NADP+  pyruvate + CO2 + NADPH (pyruvate can enter mitochondria,
carboxylated to OAA by pyruvate carboxylase)
*One molecule of NADPH is generated for each molecule of acetyl CoA that is
transferred from mitochondria to the cytoplasm.
Pentose phosphate pathway
CAC, transport of OAA from mitochondria, pentose phosphate pathway = carbon
atoms + reducing power
Glycolysis + Oxidative phosphorylation provide ATP
Synthesis: acetyl CoA + ATP + HCO3  malonyl CoA + ADP + Pi + H+ =
committed step must be regulated (acetyl CoA carboxylase)
(+) citrate – acetyl CoA and ATP are abundant
(+) insulin
(-) palmitoyl CoA – excess FA
(-) glucagon
Degradation: Malonyl CoA inhibits carnitine acyltransferase I (prevent acyl CoAs
from entering mitochondrial matrix)
Cannot be stored (like glucose/FA) nor excreted. Must be used as metabolic fuel. α-
amino group removed, carbon skeleton converted into major metabolic intermediate.
Amino group can go through urea cycle
Amino acids come from 1. digestion (diet) – low pH + enzymes:
Stomach – pepsin, Pancreas: chymotrypsin, rypsin, carboxypeptidase, elastase
2. degradation (turnover) of cellular proteins (damaged by oxidation, errors in
translation, misfolded, changing metabolic needs, unneeded and ubiquitinated)
Used to make proteins or nitrogenous compounds (nt bases)
Ornithine decarboxylases – 11 min, Hb – red blood cell, Crystalline – life of
organism
Ubiquitin: protein attached to the ε-amino group of lysine residues of proteins to be
degraded (isopeptide bond)—energy from ATP hydrolysis
E1: Ub-activating enzyme (adenylates Ub, transfers Ub to its Cys by thioester bond)
E2:Ub-conjugating enzyme (transfer Ub to its own Cys residue)
E3: Ub-protein ligase (read N-terminal, transfer Ub to Lys residue on target protein)
Half life of a cytoplasmic protein is determined by its amino-terminal residue
Stabilizing residues (t½ > 20 mins) Met, Pro
Destabilizing residues (t½ < 2 mins) Arg, Leu
Describe the fates of the peptides
derived from a ubiquinated protein
Markers for destruction
What digests the Ub-tagged proteins?
Two types of intracellular proteases
Describe proteolysis within 20S core
particle
Major organ responsible for amino acid
degradation in mammals
Describe the two steps to remove
nitrogen from AA in ____
How is glutamate dehydrogenase
regulated?
Essential cofactor for many enzymes of
aa metabolism (Dietary precursor?)
Structure of pyridoxal phosphate (PLP)
and pyridoxamine phosphate (PMP)
Reactive functional group
Transport of nitrogen from muscle to
liver
Fate of NH4+
Where is urea synthesized?
How are ammonium ions transported?
Molecules that bring nitrogen and
carbon into the urea cycle
Carbamoyl phosphate synthetase
mechanism
ATP requirement of the urea cycle
Enzymes of the urea cycle, intracellular
locations
Molecular connection between urea
cycle and citric acid cycle
Reaction catalyzed by each enzyme
Broken into amino acids that are then broken into:
1. amino group  urea cycle
2. carbon group  glucose/glycogen synthesis, cellular respiration, FA synthesis
3. stay intact for biosynthesis
Ubiquitination
Cyclin destruction boxes – aa sequences that marks cell-cycle proteins for
destruction
PEST sequence – proline, glutamic acid, serine, threonine
N terminal
The 26S proteasome = 20S catalytic unit (α2β 2) + 19S regulatory unit (binds poly-
Ub chains, ATP hydrolysis to unfold substrate, induce conformational changes in
20S to pass substrate through center)
recycles Ub
1. Sequestered proteases: lysosomal cathepsin (active at low pH), ER proteases
(degrade proteins that fold incorrectly), 2. Regulated proteases: calpain
Contains an N-terminal Thr whose –OH group is nucleophilic, attacks carbonyl
bonds in protein to form acyl-enzyme intermediate (~ chymotrypsin), MHC I
Liver, muscle also degrades branched aa (Leu, Ile, Val)
In Liver
1. α-aa + α-ketoglutarate  α ketoacid + glutamate (aminotransferase/transaminase)
2. glutamate + NAD+ + H2O α-ketoglutarate + NADH + NH4+ (glutamate dh)
Direction determined by concentration of R and P, reaction usually driven toward α-
ketoglutarate because of rapid removal of NH4+ into urea cycle
Pyridoxal phosphate (PLP) = prosthetic group of aminotransferases, derivative of
vitamin B2, has ability to tautomerize
Functional group: aldehyde (forms Schiff base with amino group of amino acids)
allows α-amino groups to be shuttled between amino acids and keto acids.
Internal  external aldimine, resonance stabilized carbanion, hydrolysis to PMP.
Muscles transport ammonium ions as alanine or glutamine.
Used to synthesize purine and pyrimidine bases
Excess excreted as NH+ in aqueous animals
Excreted as uric acid (remove 4N) in birds/reptiles, excrete more N + use less water
Excreted as urea in terrestrial vertebrates (ureotolic), secreted in solution in urine
Urea is synthesized in Liver (also major site of deamination)
Ammonium ions are transported to liver as alanine or glutamine
1 Nitrogen from aspartate, 1 Nitrogen from free ammonium
Carbon atom from HCO3-
Enzyme: carbamoyl phosphate synthase (CPS), hydrolyze 2 ATP, irreversible rxn
1. Bicarbonate +ATP  ADP + carboxyphosphate
2. carboxyphosphate + NH2  carbamic acid + Pi
3. carbamic acid + ATP  carbamoyl phosphate + ADP
1. Ornithine + carbamoyl phosphate 
citrulline (ornithine transcarbamoylase – mito
matrix)
2. citrulline (transported to cytoplasm) +
aspartate + ATP  argininosuccinate +AMP
+ Ppi (argininosuccinate synthetase)
3. argininosuccinase  arginine + fumarate
(argininosuccinase)
4. arginine + H2O  ornithine + urea

Structural/functional properties of two
isozymes of carbamoyl phosphate
synthetase in mammals
Deficiencies in different enzymes of
the urea cycle give rise to ___, outline
strategies for coping with deficiencies
in urea synthesis
How do humans catabolize carbon
atom skeletons of amino acids, name 7
major metabolic products formed
Basis for the glycogenic-ketogenic
designations of the amino acids and
classify each amino acid. Limitations of
classification
Chapter 26: Fatty acid and
Cholesterol Metabolism
Three lipid based components of
biological membranes. Biological
Significance of cholesterol
What is apo B?
Two forms
Primary site of TAG synthesis
Transported where? Function?
Major sites in cholesterol biosynthesis,
key intermediates
Synthetic paths leading __ or ___
The major regulatory enzyme in
cholesterol biosynthesis?
Conversion of mevalonate into ___
(activated ___)
Outline condensation reactions leading
from isopentenyl pyrophosphate to
squalene
Then?
(arginase)
CPS I CPS II
In mitochondrial matrix In cytoplasm
Function in urea cycle only Functions in pyrimidine biosynthesis,
first reaction in multi-subunit complex
Uses NH3 as substrate Uses glutamine as substrate
Allosterically activated by binding of Hydrolyzes side chain amide of
N-acetyl glutamate (made when aa glutamine to produce HN3 needed for
breakdown ↑s, signal to activate urea the first reaction, NH3 is not a direct
cycle substrate
* catalytic site in one isozyme = allosteric site in other*
Urea = major route of ammonium removal.
Block carbamoyl phosphate synthesis, or urea cycle  hyperammonemia (elevated
ammonium levels in the blood), may produce osmotic effects/brain swelling
- argininosuccinase deficiency: add arginine in diet, restrict total protein intake
(arginine split into urea and ornithine, reacts with carbamoyl phosphate to form
citrulline), nitrogen secreted as argininosuccinate
-carbamoyl phosphate synthetase deficiency/ornithine transcarbamoylase
deficiency (can’t use citrulline/argininosuccinate to dispose nitrogen because
formation is impaired), excess N accumulates in glycine/glutamine.
- supplement diet with benzoate + phenylacetate
Pyruvate (A, C, G, S, T, W), acetyl CoA (I, L, W), acetoacetyl CoA(L, K, P, W, Y),
α-ketoglutarate (R, Q, E, H, P), succinyl CoA (I, M, T, V), fumarate (D, P, Y),
oxaloacetate (N, D)
Ketogenic : aa degraded to acetyl CoA/acetoacetyl CoA (can be converted to ketone
bodies/fatty acids)
Glucogenic: aa degraded to pyruvate, α-ketoglutarate, succinyl CoA, fumarate, or
OAA (can be converted to PEP, then to glucose)
Phospholipids, spingolipids, cholesterol.
Cholesterol is a membrane component and precursor of signal molecules (steroids
progesterone, testosterone, estrogen, and cortisol)
Cholesterol is transported in blood by LDL, taken up by cells with LDL receptor on
surface
Protein that transports triacylglycerols and cholesterol by forming an amphipathic
spherical shell around the lipids carried in lipoprotein particles
Apo B-48: synthesized by small intestine, carries dietary fat in the form of
chylomicrons
Apo B-100: synthesized by liver, participates in the transport of lipids synthesized in
the cell
Liver, where TAGS are transported to muscle for energy conversion, adipose cells
for storage
1. In cytoplasm: acetoacetyl CoA + acetyl CoA +H2O  HMG CoA + CoA:
- mevalonate (HMG CoA reductase – integrated membrane protein in ER)
*committed step*
- acetoacetate (ketone body) + acetyl CoA
Mevalonate    isopentenyl pyrophosphate (C5) = activated isoprene
2. In ER: isomerize isopentenyl pyrophosphate ↔ dimethyl pyrophosphate
dimethyl pyrophosphate + isopentenyl pyrophosphate ↔ geranyl pyrophosphate (C10)
geranyl pyrophosphate + isopentenyl pyrophosphate ↔ farnesyl pyrophosphate (C15)
2 farnesyl pyrophosphate ↔ squalene (C30)
3. squalene activated by conversion into squalene epoxide
Squalene epoxide cyclized to lanosterol by protonation (oxidosqualene cyclase)
Lanosterol -3 methyl groups, (19 steps) = cholesterol
Sources of cholesterol
Mechanism of regulation of cholesterol
biosynthesis
3 fates of liver cholesterol
How are Cholesterol and TAG
transported in body fluids?
Macromolecular aggregates? 2 roles?
Apolipoproteins
Classes of lipoproteins, their lipid and
protein components
Lipid transport functions
Steps in delivery of cholesterol to cells
via LDL receptor.
Fates of released unesterified
cholesterol? Danger of high
concentration? Regulation of cellular
functions by the LDL pathway
Biochemical defects of LDL receptor
that result in familial
hypercholesterolemia
Approaches to reduce serum
cholesterol
Diet or de novo synthesis in liver, intestine
1. control HMG-CoA reductase activity: phosphorylation by AMP-dependent
protein kinase ↓ activity [no cholesterol/FA synthesis when ATP is low]
2. control HMG-CoA reductase concentration:
- monitor blood cholesterol: scap senses cholesterol, escorts SREBP when no
sterols are in cell from ER to Golgi, S1P and S2P releases NH2 end of
SREBP = TF, SREBP dimers bind sterol response element SRE in nucleus,
enhance transcription of HMG-CoA reductase gene
- regulate translation of HMG-CoA reductase mRNA, inhibited by cholesterol and
nonsterol compounds derived from mevalonate
- regulate HMG-CoA reductase degradation (stimulated by cholesterol, sterols, and
farnesol), has an intrinsically short half life
1. used for membrane synthesis, organ needs
2. converted to bile salts (only way to excrete cholesterol)!
3. excess cholesterol and TAG packaged into LDL and exported to blood.
Transported in the form of lipoprotein particles. Each particle has a core of
hydrophobic lipids surrounded by a shell of polar lipids/proteins. Apoproteins =
macromolecular aggregates. Two roles: solubilize hydrophobic lipids, contain cell-
targeting signals, Apolipoproteins (protein constituents of lipoprotein particles)
synthesized and secreted by liver and intestine
Chylomicrons – carry TAG, cholesterol, and lipids from intestines (~90% TAG)
*lipoprotein lipases (on blood vessel linings in muscles that use FA as fuels and
in synthesis of lipids) hydrolyze and release TAG
Chylomicron remnants – liver takes up these cholesterol-rich residues
vLDLs – mostly lipids + apo-B100 and apo-E, as they travel through blood stream,
TAG are hydrolyzed by lipoprotein lipases on capillary surfaces (muscle, adipose)
IDLs – ½ taken up by liver, ½ converted by removal of more TAG
LDLs – major carrier of blood cholesterol, core contains ~1500 esterified
cholesterol molecules surrounded by a shell of phospholipid and 1 copy of apo B-
100 (recognized by target cells)
LDL transports cholesterol to peripheral tissues to provide this compound to cells
that do not make cholesterol de novo (all except liver and intestine)
HDLs picks up cholesterol released into plasma from dying cells and membranes
undergoing turnover, acyl transferase esterifies cholesterol, returned to liver
LDL taken up by receptor-mediated endocytosis
1. LDL binds LDL receptor
2. complex invaginated to form an internal vesicle
3. LDL-containing vesicle separates form receptor, fuses with lysosomes
4. LDL degraded, cholesterol released
released unesterified cholesterol is used for membrane biosynthesis, esterified by
acyl CoA-cholesterol acyltransferase for storage in cell
disrupts membrane integrity
SREBP regulates txn of LDL receptor gene regulated, blocked by high [cholesterol]
- Familial hypercholesterolemia: genetically defective LDL receptor synthesis, cells
can’t take up LDL, high LDL and cholesterol in blood, cholesterol deposited in
various tissues (skin and tendon), oxidized LDL taken up by macrophages, form
foam cells, form atherosclerotic plaques, narrow arteries.
- block reabsorption of bile alts
- ↑ production of LDL receptors (remove cholesterol form circulation)
Key: ↓ [cholesterol ], ↑ txn of LDL receptor gene.
1. oral administration of positive polymers that bind negative bile salts to prevent
reabsorption of bile salts
2. inhibit cholesterol synthesis by inhibiting HMG CoA reductase (statins)
3. manage lipid intake in diet (egg, shrimp, etc)
Physiologic roles and general structures polar derivatives of cholesterol made in liver, stored in gall bladder, released in
of the bile salts small intestine, solubilize dietary lipids by forming micelles containing TAGs
Derivatives of Cholesterol Progesterone (made in corpus luteum) implant embryo, maintain pregnancy, ovarian cycle
Estrogen (made in ovaries) – develop 2° sexual chrs, maintain ovarian cycle
Androgens (made in testes) – develop 2° sexual chrs
Glucocorticoids (cortisol – made in adrenal cortex) – promote gluconeogenesis, ↑
degradation of fat and protein, inhibit inflammation
Farnesyl pyrophosphate – precursor for chlorophyll, Vitamin K, Coenzyme Q
Chapter 28: DNA 1. two polynucleotide chains coil to form a right-handed double helix
Features of Watson and Crick’s double 2. purine and pyrimidine bases are on inside of the helix, phosphate and deoxyribose
helical structure of DNA units are on the outside
3. A-T, G-C base pairs
Functions of free nucleotides 1. energy currency
2. Signaling molecules and regulators (cAMP, GTP/GDP)
3. activation of other molecules (UDP-glucose)
4. electron carriers (FAD/NAD+)
5. constituents of co-factors (CoA
Data Watson and Crick deduced 1. structure of bases and nucleotides
2. base pairing from ratios
3. x-ray diffraction data was diagnostic of a right handed helical structure, space
between nucleotide (3.4 Å).
4. fiber density indicated 2 molecules DNA per structural unit (double helix)
The role of deoxyribose puckering in A-DNA, C-3’ lies out of plane (C-3’ endo), bp tilt 11 degrees away from normal to
determining the structural differences the helix, phosphate bind fewer water molecules
between A-DNA and B-DNA B-DNA: C-2’ lies out of plane (C-2’ endo)
Compare structures of dsRNA and dsRNA and RNA-DNA hybrids have similar double-helical form as A-DNA
RNA-DNA hybrids with that of the A- RNA’s ribose 2’OH can’t form B helix because of steric hindrance (2’-oxygen
DNA helix would be too close to three atoms of adjacent 2’ phosphate group.) A type: 2’oxygen
projects outward, away from atoms
Major groove vs. Minor groove of B- Minor groove: pyrimidine O-2 and purine N-3
DNA Major groove: opposite side of the pair
Thymine’s methyl lies in major groove
B DNA: major groove is wider (12 vs. 6 Å) and deeper (8.5 vs 7.5 Å)
Larger groove makes it more accessible for interactions with proteins that recognize
specific DNA sequences
Structural information provided by x- X-ray diffraction studies (less-hydrated DNA fibers) = A-DNA
ray diffraction analysis of DNA fibers - not uniform, local deviations from average structure
and DNA crystals - rotation angles vary
- two bases are not perfectly coplanar, show propeller twist to enhance base stacking
Compare A, B, and Z type DNA Characteristic A B Z
Width Broadest Intermediate Narrowest
Rise/bp 2.3 Å 3.4 Å 3.8 Å
Helix diameter 25.5 Å 23.7 Å 18.4 Å
Screw sense Right Right Left
Glycosidic bond Anti Anti Alternating anti
and syn
Bp/turn of helix 11 10.4 12
Pitch/turn of helix 25.3 Å 25.4 Å 45.6 Å
Tilt of base pairs 19° 1° 9°
from normal to
helix axis
Major groove Narrow and very Wide and deep Flat
deep
Minor groove Broad and Narrow and deep Narrow and deep
 shallow

Z-DNA’s helix is what handedness? Left-handed

Shape of path traced by phosphodiester Zigzags, form adopted by short oligonucleotides that have sequences of alternating
backbone pyrimidines and purines.
Linking Number Lk = number of times that a strand of DNA winds in right handed direction around
Twisting number the helix axis
Writhing number (+) = right handed winding, (-) left handed winding
DNA topoisomer? Tw = measure of helical winding of DNA strands around each other
(+) = winding, (-) = unwinding
Wr = measure of coiling of the axis of the double helix (supercoiling)
(+) = left handed supercoiling, (-) = right handed supercoiling

Topoisomer = DNA that differ only in linking number

How does supercoiling affect the
electrophoretic and centrifugal mobility
of the molecule?
How can a negative super-helix density
facilitate the unwinding of the helix?
How is free energy of negative
supercoiling partitioned into Tw and
Wr?
Topoisomerase
Type I
Type II
Substrate, product, mechanisms
Linking number effect of
Topoisomerase I
Topoisomerase II
How can you tell the difference
between ss and dsDNA?
How do you detect specific DNA/RNA
sequences in vitro?
In vivo?
Compare structure of DNA, RNA, and
proteins
Supercoiling makes DNA molecule more compact and it moves faster in
centrifugation or electrophoresis.
Negative supercoiling introduces a torsional stress that favors unwinding of the
right-handed B-DNA double helix.
Free energy is minimized when 70% of change in Lk is expressed in Wr (everything
is bp’d so more energetically favorable) and 30% is expressed as Tw. A lowering of
Lk causes both negative supercoiling of DNA axis and unwinding of the duplex
Topoisomerase – introduce or eliminate supercoils
Type I: relax supercoiled DNA, thermodynamically favored, tyrosine cleaves one
strand of DNA, controlled rotation
Type II: utilize free energy from ATP hydrolysis to add negative supercoils to
DNA, cleave both strands of DNA, G “gate” ds segment binds enzyme, T
“transported” segment binds loosely, enzyme domain binds ATP, conformational
change cause domains to come together, cleave G segment and trap T segment,
strand joined by tyrosine-phosphodiester linkage , DNA cannot rotate, decreases
linking number by two.
Both use tyrosine to form covalent links to the polynucleotide backbone when it is
transiently broken
Topoisomerase I unwinds 1 negative supercoil per cycle:
(↑ Lk +1, Wr +1, Tw = 0)
Topoisomerase II breaks both DNA strands,
(↓ Lk -2, Wr -2, Tw = 0)
ssDNA has a higher absorbance at UV λ 260nm
Hybridization of labeled DNA to nucleic acid bound to membrane = southern
blotting (DNA), northern-blotting (RNA)
- electrophoresis on agarose gel, 2. transfer of DNA by blotting onto
nitrocellulose sheet, 3. add labeled DNA probe, 4. autoradiography shows
DNA probe
PCR amplification of DNA
In vivo: fluorescent in situ hybridization (FISH)
DNA RNA Protein
Secondary Double helical, B- Diverse – stems, α-helices, β -
structure conformation loops, bulges sheets
Tertiary structure No, supercoil Considerable, H- H bond, ionic
bonding of bonds, van der
unpaired bases Waals
Structural Limited Considerable Enormous
plasticity deviations from B

Key features and reactions catalyzed by
DNA polymerases
What are the common structures and
evolutionary relationships among DNA
polymerases?
What are the two roles of Mg2+ in the
reaction catalyzed by DNA
polymerases?
What orients the metal ions?
How does a DNA polymerase select the
correct incoming dNTP substrate?
All replicative DNA polymerases are
associated with _____
Klenow Fragment
Enzymes that form and remove RNA
primers from the genome.
Substrates and reaction mechanisms of
DNA ligase
Similarities and Differences between
bidirectional replication of linear vs.
circular DNA?
Reactions and movements of DNA
strands that occur during replication/
define replication fork
How do helicases separate the strands
of duplex DNA?
Define Processivity
Role of β2 subunit of DNA pol III
Role of γ
Physical stability High Moderate Low
Chemical stability High (bases in Moderate Variable
center) (susceptible to alkaline
hydrolysis)
Chemical variety Low – 4 bases Low – 4 bases, High—20 aa
modified
Chemical Very low – bping, Moderate: RNAs High: catalyze lots
reactivity bases and backbone can catalyze limited of rxns, bind ions,
can be bound by rxns like metabolites, and
proteins transesterifications macromolecules
DNA polymerase catalyzes the formation of polynucleotide chains, adds nt to 3’ end
of polynucleotide chain, catalyzes the attack by 3’-OH to the α phosphoryl group of
the nucleoside triphosphate. Needs a primer to initiate
Finger and thumb – wrap around DNA and hold it across enzyme’s active site
Palm: active site
Two metal ions – catalyze the polymerase reaction
1. activate the 3’-OH of the primer, facilitates nucleophilic attack of α-phosphate
group of dNTP substrate. Two ions help stabilize the negative charge that
accumulates on the pentacoordinate transition state.
2. stabilize the negative charge on the pyrophosphate product.
Conserved Asp
Shape complementarily – nucleotide with similar shape, but different hydrogen
bonds, can still be incorporated
1. DNA polymerase forms hydrogen-bonds with the minor groove side of the bp in
the active site to check for proper spacing
2. DNA polymerase close down around incoming dNTP, finger domain forms a tight
pocket that ensures proper shape of base pair.
3’5’ exonuclease activity
Contains polymerase unit and 3’5’ exonuclease activity
Primase forms RNA for initiation
DNA pol I erases primer with 5’  3’ exonuclease and fills in gaps on lagging
strand with polymerase activities
Seals the nicks after replacement of RNA primers
Seals breaks in double-stranded DNA molecules, cannot link two molecules of
ssDNA or circularize ssDNA
Similarity: origin of replication, synthesis the same
Difference: nature of products, circular DNA becomes intertwined and must be
separated by topoisomerase II, end problem of linear DNA
DNA pol III synthesizes the leading and lagging strand simultaneously at the
replication fork. Primase synthesizes RNA primer, duplex DNA ahead is unwound
by DnaB (helicase), SSB bind unwound strand, Topoisomerase II introduces
negative supercoils to prevent topological crisis
A1 (P-loop NTPase fold) and B1 bind ssDNA, ATP binds and the cleft closes and
domain A1 slides along the DNA. On ATP hydrolysis, the cleft opens up, pulling
DNA from B1 toward domain A1.
Processivity – the ability of an enzyme to catalyze many consecutive reactions
without releasing its substrate
β2 unit keeps the polymerase associated with the DNA double helix = sliding clamp
γ binds ATP, induces conformational change, binds and opens β ring
ATP hydrolyzed and β ring closes and clamp loading complex released
Distinctive features of DNA pol III
holoenzyme
How does an asymmetric dimer of the
enzyme coordinate the synthesis of
leading and lagging strands of the
daughter duplexes?
___ model
Role of DNA pol I and DNA ligase in
replication
Function and features of the nucleotide
sequence of oriC
Bidirectional replication initiation in E.
coli?
Proteins that interact with DNA in
oriC, reactions catalyzed and functions
served
Problem arising from DNA length and
cell cycle in eukaryotes. Rate of
replication? Solutions?
In Eukaryotes, DNA synthesis is ___
Replication licensing ensures ____
Replication Initiation in Eukaryotes
Licensing factors accumulate only
during ___
Ways to get rid of licensing factors?
Why important to eliminate factors?
Define Telomeres
How does telomerase make DNA of
defined sequences in the absence of a
DNA template?
Polymerase core: αεθ
Sliding clamp: β2
clamp loader: γτ2δδ’
DnaA: binds oriC, forms hexamer
DnaB: helicase
DnaC: helicase loader protein
DnaG: primase
Lagging strand looped out so it passes through polymerase site in the same direction
as the leading strand template. Pol I lets go of the lagging-strand template after
adding 1000 nts by releasing the sliding clamp. A new loop is formed, a sliding
clamp is added, and primase is synthesized in short stretches. Trombone model –
loop lengthens and shortens.
Gap between lagging strands filled in by pol I. (has 5’  3’ exonuclease activity)
DNA ligase connects the fragments.
Origin of replication has 5 copies of a sequence that are binding sites for DnaA +
tandem 13-b AT sequence repeats.
To start replication:
1. DnaA binds to 5 sites in oriC, come together to form a cyclic hexamer
(dissociates at beginning of initiation to prevent additional rounds of replication
from beginning), DNA wraps around
2. ssDNA are exposed in the pre-priming complex. DnaB (helicase) loaded by
DnaC, unwinds DNA, SSBP traps ssDNA. DnaG (primase) inserts RNA primer
3. Polymerase holoenzyme: DNA pol III holoenzyme assembles, initiated by
interactions between DnaB and sliding clamp subunit. ATP hydrolysis within
DNA subunits breaks up assembly, signal the initiation of DNA replication
4.6 million bp, humans have 6 billion bps. 1 vs 46 chromosomes, circular vs. linear
chromosome, rate – 60-90 nt/sec. 30,000 replication origins (each origin is the
starting site for a replication unit, replicons)
Telomeres: prevent loss of information because polymerase can only act in 5’ 3’
direction
DNA synthesis is only takes place during S phase of cell cycle
Replication licensing ensures that the genome is replicated once, and only once, per
cycle
1. assemble ORC (6 proteins ~DnaA)
2. Cdc6 + cdt1 recruit Mcm2-7 (helicase) = Licensing factors + Replication protein
A (SSBPs)
3. polymerase switching: Pol I ~ α initiates replication, Pol III ~ δ = processive pol
4. after polymerase α adds 20 dNTPs to primer, replication factor C (RFC)
displaces pol α. RFC attracts sliding clamp (PCNA – proliferating cell nuclear
antigen ~ β2)
PCNA + DNA pol δ makes the enzyme highly processive, continue to replicate until
adjacent replicons fuse
G1
After phosphorylation, Mcm – nuclear export
Phosphorylated cdc6 – degraded
Cdt1 – sequestered by binding Genin (present from s M phases, then degraded)
To prevent setting up new origin of replication
Telomeres: specialized structures at the ends of linear chromosomes.
(Tandem repeats of six-nt sequence at 3’ end of DNA, the single-stranded segment
of G-rich strand extends from the end of the telomere. Ss region invades the duplex
and forms a large duplex loop to protect genomic ends.)
Telomerase contains an RNA molecule that serves as a template for elongation for
the G-rich strand, reverse transcriptase
Common types of mutations in DNA
Involvement and limitations of error-
prone DNA polymerases.
Trinucleotide-repeat expansions
How do they relate to Huntington
disease?
General strategies for repairing
damaged DNA
How are mismatches, pyrimidine
dimers, alkylated bases repaired?
Double strand breaks induced by
Why are they potentially dangerous?
Repaired by
Why are low rates of mutation
essential?
How are low mutation rates
maintained?
High fidelity in DNA replication is the
result of
Difference between base-excision
repair and nucleotide-excision repair
Chapter 29: RNA metabolism
Subunit composition of RNA
polymerase of E. coli for holoenzyme +
core enzyme
Describe function of each subunit
Comparison of DNA and RNA
synthesis
1. formation of pyrimidine dimers induced by UV irradiation
2. depurination and deamination
3. spontaneous alterations
4. exposure to chemical mutagens – nonstandard base pairing
Base analog mutagens (5-BU, 2-AP incorporated into DNA, form transient
tautomers)
Nitrous acid deaminates bases
DNA polymerases that can replicate DNA across many lesions, allow the
completion of a draft sequence of the genome that can be partially repaired by DNA-
repair processes, DNA recombination
An autosomal dominant neurological disorder, mutated gene creates glutamine
residues, increased number of trinucleotide sequence. Polyglutamine stretches
aggregate
1. Proofreading – growing polynucleotide chain leaves the polymerase site,
migrates to nuclease active site, remove incorrect bases
2. mismatch repair – 2 proteins (one detects mismatch, other recruits endonuclease
to cleave DNA) MutS recognizes mismatch, MutL calls MutH to cleave backbone.
(newly synthesized DNA is unmethylated)
Ionizing radiation, oxidizing agents, errors in replication
Potentially dangerous because there is no intact template strand to guide repair (as I
NER & BER)
They are repaired by nonhomologous end joining, homologous end joining.
In germline to maintain thee species
In somatic cells to prevent individual from developing cancer, harmful mutations
1. accuracy during DNA synthesis
2. post-replicative mechanisms that repair most damage
1. Double checking of base-pairing geometry before covalent addition of a
nucleotide (higher affinity for correct nt because of bping with template)
2. remove first new nts synthesized that are most likely to be mispaired
3. exonucleolytic proofreading – mispairing induces change from active site to
exonuclease site (kinetic proofreading – delay after mistake allows spontaneous
melting and released 3’ end to contact exonuclease site, excises mismatch)
Base excision repair – mediated by DNA glycosylases (enzyme flips out nucleotide
to evaluate each bp, removal of sugar phosphate residues by cleaving glycosidic
bond) DNA backbone is intact, base is missing AP site (ex: AlkA enzyme)
Nucleotide-excision repair – excise pyrimidine dimer, entire nucleotide including
backbone. The distortion of the B-helix is recognized by the multienzyme repair
complex.
Holoenzyme: α2β β ’σ Core enzyme: α2β β ’
α – binds regulatory proteins and sequences
β - binds NTPs and catalyzes bond formation
β ’ – binds the DNA template
σ70 – recognizes the promoter (-35 and -10 region) by scanning sequence in 1
direction and initiates synthesis, only binds DNA when associated with core
enzyme, otherwise has an autoinhibitory domain to block DNA-binding surfaces
Similarities:
1. initiate at specific site in genome (origin – replication, promoter – transcription)
2. produce new nt chains by synthesis in the 53’ direction
3. driven by hydrolysis of pyrophosphate after incorporation of dNMP or NMP from
dNTP or NTP precursors
Differences:
1. only specific segments of genome are copied into RNA
2. only one of two strands serves as template
3. RNA polymerases initiate synthesis de novo

How do RNA polymerases catalyze
nucleophilic attack?
Strong vs. Weak promoters
Three stages of RNA synthesis and
functions of RNA polymerase in
processes
Eukaryotic differences in transcription
How does σ factor enable RNA
polymerase to recognize promoters?
Difference between σ70 and σ32?
How was topoisomerase I used to
determine the number of promoter
DNA base pairs that are unwound upon
binding of RNA polymerase
 how does negative supercoiling
relate to promoter efficiency?
Two strands of DNA twist around the
helica xis once very ___ bps
Closed promoter vs. Open promoter
complexes
How does topoisomerase II (Aka: ___)
facilitate transcription?
Detail the de novo initiation of chain
growth by RNA polymerase, name
unusual initiating ribonucleosides
2 steps to transcriptional Elongation
What is the transcription bubble?
Number of base pairs in RNA-DNA
hybrid.
Rate of RNA chain elongation (nt’s
added? Distance on the template
traversed by RNA polymerase?) vs
4. transcription has higher degree of regulation, genes only transcribed under
specific conditions
Nucleophilic attack by terminal 3’ OH in nascent RNA chain on the α-phosphate
group of the incoming NTP
strong promoter: have close match in -10 and 35 to consensus sequence, can be
transcribed every 2s (promoters encoding rRNAs)
weak promoters: differ at multiple sites within -10 and -35 sequences, or space
between them (17 bp is optimal)
1. promoter binding and initiation
2. elongation of nascent RNA transcript
3. termination at end of gene
- regulated by TF binding (control promoter activity)
- enhancer sequences imitate transcription
- modified transcripts: 5’ cap and 3’ poly A tail
RNA polymerase and σ factor slides along DNA, specifically binds to promoter site
on template DNA. Released after RNA chain 9-10 nts long (acts catalytically)
Σ70 recognizes consensus sequence
When temperatures are raised suddenly, E. coli synthesizes σ32, which recognizes
promoters of heat-shock genes (promoters exhibit -10 sequences that are different
from standard promoters, leads to synthesis of protective proteins). Σ plays a role in
determining where RNA polymerase initiates transcription (starved, heat, etc)
Experiment: analyze the supercoiling of circular duplex DNA exposed to varying
amounts of RNA polymerase.
Topo I added to relax (Wr +1) part of circular DNA not in contact with polymerase
molecules. Remove bound protein and analyze DNA samples by gel
electrophoresis.
↑ # of RNA polymerase bound/template DNA led to ↑ in degree of negative
supercoiling.
∴ RNA polymerase unwinds DNA by (-Tw or –Wr: supercoil)
Each bound polymerase molecules unwinds a 17 bp segment of DNA, which
corresponds to 1.6 turns of B-DNA helix.
10.4 bps
Closed – DNA is double helical
Open – DNA segment is unwound
Aka DNA gyrase facilitates transcription by introducing negative supercoils.
Negative supercoiling in circular DNA favors transcription because it favors
unwinding. (Exception: negative supercoiling of promoter site for topoII gives
negative feedback, decreases efficiency, prevent excessive supercoiling).
Unwind the template to form an open promoter complex
Very tight association of enzyme with template
First base: pppG or pppA
1. promoter clearance : after 5-10 nt are added to nascent RNA, σ dissociates from
the complex (lose contact with promoter, affinity of the core enzyme for template
increases by a factor of 104 – nonspecific to sequence)
2. Processive elongation without accessory proteins. RNA polymerase acts a
molecular motor on the DNA – produces greater forces than myosin/kinesin
Region containing RNA polymerase, RNA-DNA hybrid
8 bp, 1 bp of DNA unwound in elongation phase (constant length of unwound region
means DNA is rewound at the same rate as unwound)
RNA synthesis: 50 nt/sec
Distance traveled: 170 Å/sec
DNA synthesis: 800 nt/sec
DNA rate
Termination signals in RNA
ρ -dependent vs. ρ -independent
transcription termination. Outline
mechanism of ρ protein and role of
ATP hydrolysis in function
ρ structure similar to ____
Compare specific subunits
Mechanisms of inhibition of
transcription by rifampicin and
actinomycin D
Processing and modification of the
precursors of mRNA, rRNA, and tRNA
3 Regulatory differences?
Spatial/temporal differences in
transcription and translation between
prokaryotes and eukaryotes.
Difference in mRNA processing in
prokaryotes and eukaryotes
3 RNA polymerases of eukaryotes.
Location in cell. RNA they synthesize,
effect of α amanitin
Transcribed regions of DNA templates contain stop signals (GC palindrome hairpin
loop followed by 4+ uracils. RNA polymerase pauses after synthesizing hairpin,
weakly bound nascent RNA dissociates from DNA template, DNA template
renatures to close transcription bubble.
ρ protein helps to terminate transcription of some genes (ex: 23S RNA  10S, 13S
and 17S RNA)
ρ hydrolyzes ATP n the presence of ssRNA, but not in the presence of DNA or
duplex RNA. Hexameric ρ binds ssRNA (72 nt) so that RNA passes through center
of structure, ATPase activity enables protein to pull the nascent RNA while pursuing
RNA polymerase. When it catches up to RNA polymerase at transcription bubble,
ρ acts as a helicase and breaks RNA-DNA hybrid helix
ATP synthase, hexameric
ssRNA plays role of γ subunit
Rifampicin is an antibiotic that interferes with the formation of the first few
phosphodiester bonds in the RNA chain by blocking the channel into which the
RNA-DNA hybrid generated by enzyme must pass. After initiation, RNA-DNA
hybrid in the enzyme prevents antibiotic blocking. Bacterial RNA polymerases
conserved, rifampicin useful against tuberculosis.
Actinomycin D inhibits transcription by binding tightly and specifically to double-
helical DNA, prevent effective template for RNA synthesis. Intercalcation –
actinomycin slips in between neighboring bps in DNA, inhibit rapid cancer growth
Prokaryotic mRNA undergoes little modification
In E. Coli, three rRNAs and a tRNA are excised from one primary RNA transcript
with spacer regions.
Ribonuclease P (Rnase P) generates the correct 5’ terminus of all tRNA molecules.
Addition of CCA sequence at the 3’ end of all tRNAs
Rnase III excises 5S, 15S, 23S rRNA precursors by cleaving double-helical hairpin
regions at specific sites
Modification of bases and ribose units of rRNAs (methylation = greater structural
and functional versatility)
3 characteristics of gene expression: nuclear membrane compartmentalization,
transcriptional regulation, RNA processing
Prokaryotes Eukaryotes
Translation begins as transcription is Transcription and translation take place
till taking place in different cellular compartments,
transcription takes place in nucleus,
translation outside in cytoplasm
3 promoter elements: -10, -35, UP Elements that regulate transcription can
elements be bound at a variety of locations in
DNA up or down stream, long
distances
Little mRNA modification 5’ cap, 3’ poly(A), splicing
Type Location Cellular Effects of α-
Transcript amanitin
I Nucleolus 18S, 5.8S, and Insensitive
28S rRNA
II Nucleoplasm mRNA precursors Strongly inhibited
and snRNA
III Nucleoplasm tRNA and 5S Inhibited by high

What unique feature does RNA
polymerase II contain?
Why is α-amanitin toxic? Can does it
differentiate the three eukaryotic RNA
polymerases?
Sequence elements of eukaryotic
promoters. Contrast nucleotide
sequences and locations of the TAT
box of eukaryotes and the -10 sequence
of prokaryotes
TATA-box binding protein role?
Transcription factors, other DNA-
binding regulatory proteins that direct
RNA polymerase to specific genes
TBP mechanism
Nucleosome
What marks transition from initiation to
elongation in RNA synthesis?
Intragenic regulators of RNApII
Properties of enhancer sequences
tRNA modifications in eukaryotes
Structure of 5’ end of eukaryotic
mRNA, purpose?
How does mRNA produce poly(A)
tail?
RNA editing
Examples of effects on gene expression
Splicing of eukaryotic mRNA
Consensus sequence
Splice site junctions
Other nt sequence elements in process
Chemistry of splicing process
rRNA concentrations
Carboxyl-terminal domain (CTD) - contains YSPTSPS consensus sequence, activity
is regulated by phosphorylation on Serine residues of CTD
Α-amanitin binds tightly to RNA pol II (small Kd = 10 nM), blocks the elongation
phase of RNA synthesis.
RNA pol I: promoter sequences are located in stretches of DNA separating the 3
rRNA genes. Ribosomal initiator element – rInr (TATA-like sequences at start
site), and upstream promoter element – UPE (150 to 200 bp from start site)
RNA pol II: enhancer elements that are more than 1 kb from start site
RNA pol III: promoters are within the transcribed sequence, downstream of start
site
- active in assembling TFII transcription complexes, between -30 and -100 position,
- Initiator Element (Inr): sequences found at transcriptional start site (-3 to 5)
- Downstream core promoter element (DPE): (+28 to +32)
TFIID (includes TBP that recognizes TATA box) begins initiation by binding to
TATA box -- binds with 105 higher affinity
TBP provides docking site for TFIIA, TFIIB, then TFIIF, RNA po II, TFIIE, and
TFIIH = basal transcription apparatus
TATA box of DNA binds to concave surface of TBP (Saddle-shaped protein),
binding induces conformational changes in DNA, double helix unwound by
widening of minor groove, enables extensive contacts with antiparallel β strands on
the concave side of TBP 1. DNA is bent, 2. unwound
Repeating units of chromatin (the complex of DNA and protein that makes up
chromosomes.) – DNA + 4 pairs of histones
TFIIH phosphorylates CTD, stabilizes transcription elongation by RNA pol II and
recruits RNA-processing enzymes that act during elongation
Sp1 (stimulatory protein 1) – sequence-specific transcriptional activator
HSF – activated in response to elevated temperature
Enhancers – cis-acting sequences that stimulate transcription several thousand base
pairs away, have no promoter activity of their own, can be up, down, or in the
middle of a transcribed gene.
5’ leader cleaved by Rnase P, 3’ trailer is removed, CCA is added, base and ribose
moieties modified, pre-tRNAs are spliced by an endonuclease and a ligase to remove
an introns
Immediate, a phosphoryl group is released by hydrolysis. The diphosphate 5’ end
attacks the α-phosphorus atom of GTP to form a 5’-5’ triphosphate linkage “cap.”
Protect 5’ end from phosphatases and nucleases, enhance translation
Endonuclease cleaves AAUAAA sequence in primary transcripts (with important
context), poly (A) polymerase adds 250 adenylate residues
Change in nucleotide sequence of RNA after transcription by processes other than
RNA splicing. Two forms of Apo B (apo B-100, Apo B-48). Cytidine deaminated to
uridine, deaminase only in small intestine
Excising of introns and linking of exons, two transesterification reactions
Introns begins with GU and ends with AG
3’ end of introns: 10 pyrimidines, C, then AG
Branch site – 20-50 nt upstream of 3’ splice site, contains nucleophilic 2’-OH
1. cleavage of phosphodiester bond between the upstream exon and the 5’ end of the
introns. Attacking group = 2’0OH group of adenylate residue in branch site to form
2’-5’ phosphodiester bond between A residue and 5’ terminal of introns
(transesterification) lariat formed

Compare number of phosphodiester
bonds broken and formed during
mRNA splicing
Spliceosome, detail structure and
catalytic involvement of small nuclear
ribonucleoprotein particles in mRNA
splicing
Role of snRNPs
Ways in which transcription and
translation are coupled in eukaryotes,
describe role of carboxyl-terminal
domain (CTD) of RNA polymerase II
Ribonuclease P
Reactions during self-splicing
conversion of rRNA precursor from
Tetrahymena into mature rRNA
Describe the role of proteins in the
spliceosome
2 pieces of evidence that U6 snRNA
catalyzes splicing
Chapter 30: Protein Synthesis
Describe tRNAs
Ribosomes
Links AA to each tRNA
Driven by _____
Common features of all tRNAs
2. 3’-OH terminus of exon 1 attacks phosphodiester bond between introns and exon
2. Exons join and introns is released as a lariat. (transesterification)
Same before and after, no energy source needed
Phosphodiester bond between exon 1 and 5’ introns broken
Phosphodiester bond between 2’OH and 5’ introns formed
Phosphodiester bond between exon 2 and 3’ introns broken
Phosphodiester bond between exon 1 and exon 2 formed
Small nuclear RNAs (snRNAs) – have less than 300 nucleotides, located in nucleus
Ex: U1, U2, U4, U5, U6 – essential for splicing mRNA precursors
Small nuclear ribonucleoproteins (snRNPs)
Spliceosome = assemblies of snRNPs, splicing factors, and mRNA precursor
U1: binds the 5’ splice site and then the 3’ splice site
U2: binds the branch site and forms part of the catalytic center (binding requires
ATP hydrolysis)
U5: binds the 5’ splice site
U4: masks the catalytic activity of U6
U6: disengages from U4 and base pairs with U2, displaces U1 @ 5’ end of introns,
catalyzes splicing with U2 (catalytic center)
2nd transesterification: U5 helps 3’-OH grp of exon 1 positioned to nucleophilically
attack 3’ splice site. U2, U4, U5 bound to excised lariat introns released to complete
splicing reaction
One of the serines must be phosphorylated in CTD, which is controlled by kinases
and phosphatases. Phosphorylation state helps CTD recruit:
1. capping enzyme, 2. components of the splicing machinery, 3. an endonuclease
that cleaves the transcript at the poly(A) addition site, creating a free 3’OH group for
3’ adenylation
Catalyzes the maturation of tRNA by endonucleolytic cleavage of nucleotides from
5’ end of precursor
1. add guanosine nucleotide can serve as energy source or an attacking group that
attacks 5’ end of introns
2. exon 1’s free 3’-OH attacks 3’ splice site = second transesterification to join the
two exons, release intron
1. Package pre-mRNA or snRNAs (hnRNP, snRNP proteins)
2. Facilitate snRNA-mRNA interactions
3. Catalyze RNA rearrangements (RNA helicases)
4. Regulate splicing
1. coordination of Mg2+ by U6 snRNA is essential for splicing
2. U2/U6 snRNA complex synthesized in vitro can bind to a synthetic RNA with a
branch point sequence and catalyze attack of the 2’OH of the branch point A on a
specific phosphodiester bond of U6 snRNA
Adapter molecules that bind to a specific codon, brings with it an aa for
incorporation into the polypeptide chain
Contains certain modified nucleosides, half nucleosides are base-paired helical
regions, contain fewer than 100 ribonucleosides
Complexes with three large RNA molecules and 50 proteins. Ribozyme = RNA
component plays the most fundamental role
Aminoacyl-tRNA synthase, driven by ATP cleavage
1. 73-93 ribonucleotides
2. 7-15 unusual bases (methylated/demethylated, methylation = hydrophobic
interactions with syntheses and ribosomal proteins)
3. ½ nts bp’d to form a double helices

Why tRNA molecules have common
structural features? Unique?
Bonds formed by aminoacyl-tRNA
synthetase reaction
Transpeptidation is ___, driven by __
∆ G°‘ of reaction catalyzed by the
aminoacyl-tRNA synthetases is
tRNA structure (det by ___)
Two-step reaction sequence of
aminoacyl-tRNA synthetases
Where are the high-energy phosphate
bonds that are consumed in the overall
reaction?
Describe the mechanisms of amino acid
selection and proofreading that
contribute to overall accuracy of
peptide
Difference in class I and class II
aminoacyl-tRNA synthetases
3D structure of a prokaryotic ribosome
RNA percentage, RNA and protein
subunits, binding sites, mRNA bound
within ___ subunit
Shine-Dalgarno sequence
2 interactions hat determine where
protein synthesis starts
Protein synthesis in bacteria starts with
How does polypeptide chain escape
ribosome?
Describe initiation of Protein synthesis
5 groups not bp’d (help with unique distinguishing), 3’ CCA terminal region
(acceptor stem), TψC loop, DHU loop (dihydrouracil), anticodon loop
4. 5’ end of tRNA is phosphorylated, 4’ terminal = pG
5. activated aa attached to –OH group of adenosine residue, located at 3’-CCA
component of the acceptor stem
6. anticodon near center of sequences
Common reactions: ribosome, Efs
Different: activating enzyme
- a mixed anhydride bond is formed to AMP
- acyl ester link to 3’ or 2’ –OH of tRNA
Spontaneous, driven by formation of peptide bond that is stronger than the ester
bond
~0 kcal/mol because free energy of hydrolysis of ester bond of aminoacyl-tRNA =
free energy of hydrolysis of phosphate bond ATP  AMP + Ppi
Determined by x-ray crystallography
L-shaped
A form DNA (4 helical regions form 2 db helix)
Nonhelical region = H-bond interactions
CCA terminus has aa-attachment site, SS region: change conformations during aa
activation and protein synthesis
1. AA + ATP ↔ aminoacyl adenylate + Ppi (mixed anhydride, COOH group
of aa linked to π group of AMP)
2. aminoacyl-AMP + tRNA ↔ aminoacyl-tRNA + AMP
 2 ATP consumed to synthesize aminoacyl0tRNA
1. acylation site eliminates Aas that are too big (won’t fit in)
2. hydrolytic site eliminates Aas that are too small (editing site removes AA by
hydrolysis if AA fits)
I: acylates the 2’-OH group of terminal adenosine of tRNA
CCA arm = hairpin conformation, monomeric
II: acylates the 3’OH group of terminal adenosine of tRNA
CCA arm = helical conformation, dimeric
Ribonucleoprotein made of small (30S) and large (50S) subunits
RNA = 2/3 mass of ribosome
3rRNAs 5S, 16S, 23S, folded into structure with short duplex regions
Have 3 tRNA binding sites that bridge the 30S and 50S subunits
mRNA bound within 30S subunit
Initiator region of purine-rich sequence with 10 nt on 5’ side of initiator codon in
mRNA of prokaryotes (bp’s with 3’ end of 16S rRNA of 30S subunit contains a
sequence that bp’s with purine rich initiator site)
1. pairing of mRNA bases with 3’ end of 16S rRNA
2. pairing of initiator codon on mRNA with anticodon of an initiator tRNA molecule
N-formyl methionine
Through tunnel in ribosome
1. 30S subunit and mRNA bind through Shine0Dalgarno sequence
2. initiator tRNA formylmethionine binds initiator AUG
3. 50S subunit binds 30S subunit – complete 0S subunit

How does peptidyl transferase center
help formation of peptide?
Chrs of peptidyl transferase center
Initiation factors
Name and describe three Efs
Release Factors
Differences between Eukaryotic and
prokaryotic translation initiation
4. Initiator tRNA in P site on ribosome
5. charged tRNA with anticodon complementary to codon in A site binds
6. peptide bond formation: Metf transferred to Aaa site, thermodynamically
spontaneous reaction, catalyzed by site on 23S rRNA (peptidyl transferase center),
amino group of aminoacyl-tRNA in A site attacks ester linkage between initiator
tRNA and Metf
7. P site tRNA = uncharged, protein enzyme EF-G translocates mRNA so next
codon shifted to A site, hydrolyze GTP
1. forming a nucleophilic NH2 group on A site aminoacyl-tRNA
2. Stabilize Th intermediate
Only rRNA groups in the peptidyl transferase center: nearest protein group is 18Å distant
IF1: complex with 30S ribosome to prevent 50S from joining without mRNA,
IF3: complex with 30S ribosome, recognizes fmet-tRNAf bt not other tRNAs
IF2: G protein family binds GTP, conformation change associates with met-tRNAf
binds mRNA, initiator tRNA, stimulates association with 50S subunit = 70S
initiation complex
EF-G: enzyme that translocates Aas (mRNA moves through ribosome by 1 codon
distance) and hydrolyzes GTP
GTP hydrolysis induces conformational in EF-G, forcing tail deeper into A site, and
hence complete shift of peptidyl-tRNA into P site and of free tRNA into E site for
release
EF-Tu: (G protein) uses GTP to bind aminoacyl tRNA and bind ribosome, two
functions:
1. protect ester linkage in aminoacyl-tRNA from hydrolysis
2. GTP  GDP hydrolysis when appropriate complex between EF-TU-
aminoacyl-tRNA complex and ribosome formed (no codon-anticodon = no
hydrolysis, no tRNA transfer), ∆ G of GTP hydrolysis contributes to
fidelity of protein synthesis, release EF-Tu from ribosome
*doesn’t recognize fmet-tRNAf, recognizes Met-tRNAm and other tRNAs (opposite
of IF2)
EF-Ts: induces dissociation of GDP from EF-Tu (GTP binds Ef-Tu and EF-Ts is
released)
RF3: mediates reaction between RF ½ and ribosome (similar to G protein Ef-Tu)
RF 1 UAA, UAG, RF 2: UAA, UGA: recognize codons and promote release of
completed protein from the last tRNA
EF-G and RRF dissociates complex
Chr Euk Pro
Ribosomes 60S + 40S = 80S 50S + 30S = 70S
initiator tRNA normal Met-tRNA formylMet-tRNAf
Initiation 40S ribosome w/ bound Shine Dalgarno
Met-tRNA attaches cap sequence, purine rich,
of 5’ end of euk. mRNA, attaches to 16S RNA
scans to 1st AUG,
helicase
# Start sites 1 start site Many start sites
5’ end Capped: mRNA circular: eIF Accessible to proteins
-4E binds cap, eIF-4G binds 2
polyA binding proteins
(PABPI)
GTP form delivers EF1 α Ef-tu
aminoacyl tRNA to A site
Catalyzes exchange of EF1 β γ EF-Ts
GTP for bound GDP
GTP driven translocation EF2 EF-G
Release factor(s) eRF1 RF 1, 2, 3

What kind of amino acids can be used
for peptide bond formation
Ionisine and purpose
Explain mechanism behind wobble
3 major proteins cleaved
Describe process by which proteins
leave cell
4 components of ribosome
translocation to ER
Mechanisms of ribosome catalysis
Maximal rate of each ribosome
Error rate
Binding energy of codon: anticodon
interaction
A kinetic Proofreading mechanism for
selecting a correct codon-anticodon
interaction
Cross-pathway Comparisons
Describe acyl adenylate intermediates
in two different pathways
Flexible arms
Carboxylases
Prevents reassociation on eIF-3 IF3
ribosome subunits
without initiator complex
L-Amino acids
Deaminated adenosine, maximizes number of codons that can be read by a tRNA
molecule, can base pair with U, C, or A
First two bases checked more heavily: 16S RNA in 30S subunit forms H bonds on
amino-groove with correctly formed bps of codon-anticodon duplex interactions that
check first two positions
1. secretory proteins – exported, lysosomal protein, proteins spanning plasma
membrane
Start translation on free ribosome, halts, directed to cytoplasm side of ER, ribosome
docks on membrane, protein synthesis begins again, during translation, polypeptides
goes through membrane  lumen of ER
1. signal sequence: 9-12 hydrophobic aa, near amino terminus
2. signal recognition particle (SRP): stop EF-binding site, halt protein synthesis
3. SRP Receptor (SR) – SRP-ribosome diffuse to ER, binds SR
4. Translocon – translocation machinery, integral and peripheral membrane
proteins, protein conducting channel (opens when ribosome binds, resumes protein
synthesis), polypeptide goes to lumen of ER
Induced fit = conformational change upon binding correct A site substrate: important
to exclude H2O
Position effects = Entropy trap
Transition state stabilization
~15 codons/sec
~ 10-4 per residue
Binding energy of codon: anticodon interaction is ~12 kJ/mol, corresponding to an
affinity constant of about 100. Therefore, codon: anti-codon interactions should have
an error rate on the order of 10-2 per residue.
- rate of productive transpeptidation, initiated by dissociation of Ef-Tu-GDP is
independent of which tRNA is bound
- rate of amino-acyl tRNA release, while retaining EF-Tu-GDP is larger when a non-
cognate compared to the cognate tRNA is bound.
∴ Incorrect amino-acyl tRNA is more likely to be released before transpeptidation,
proofreading depends on differences in the rate of a reaction when the correct vs.
incorrect substrate is bound to the ribosome.
FA activation: acceptor of acyl group = CoA
AA activation: acceptor of acyl group = tRNA
Both: irreversible by hydrolysis of pyrophosphate
Protein synthesis: flexible short stretch of polynucleotides in tRNA synthases edit
AA linkage without dissociation
Carboxylases: pyruvate carboxylase has a long flexible arm that allows prosthetic
group to rotate from one active site of enzyme to other (ATP-bicarbonate site to
pyruvate site -- gluconeogenesis), Propionyl carboxylase (degradation of odd chain
FAs converted to succinyl CoA, enters CAC), acetyl CoA carboxylase (FA
synthesis),
Pyruvate  Acetyl CoA: E2 transacetylase has flexible lipoamide cofactor attached
to lysine residue, carries substrate from active site to active side
An enzyme that catalyzes a carboxylation or decarboxylation reaction.
Acetyl CoA  malonyl CoA (acetyl CoA carboxylase): FA synthesis
Propionyl CoA  Succinyl CoA (Propionyl CoA carboxylase): after odd chain FA
degradation, conversion to succinyl CoA before entering CAC
 Pyruvate  OAA (pyruvate carboxylase) gluconeogenesis

Biotin activates CO2 group, covalently attached to ε amino of Lys

Carboxy-biotin intermediate (CO2-biotin-enzyme) formed by hydrolyzing ATP
Activated CO2 transferred to acetyl CoA, pyruvate
G proteins G proteins bind guanyl nucleotides, stimulates adenylate cyclase activity, increases
concentration of cAMP
EF-Ts catalyzes exchange for EF-Tu activated receptor (GDP  GTP)
Heterotrimeric G protein in signaling pathways
Error Frequencies: DNA, RNA, protein DNA: 10-10: 3’5’ exonuclease
RNA: 10-5: backtracking stimulated by accessory protein allows mistakes to be
Repair mechanism corrected
Protein: 10-4: editing site from aminoacyl synthase, EF-Tu specific transport of
amino acyls
Activated complexes: E1 enzyme activates ubiquitin by adenylation (hydrolyzes ATP  Ppi)
Activation part leaves FA activation, AA degradation (activation by adenylation) – pg. 622 + 862
Maintains whole Activating group is released from growing chain: fatty acid synthesis, cholesterol
synthesis, protein synthesis
Activating group is released from incoming unit as it is added to growing chain:
Glycogen synthesis (UDP-glucose), DNA synthesis (dNTP), RNA synthesis
Ub activated by E1 by adenylation before transfer to protein marked for destruction
UDP-glucose – activated glucose
ATP – activated form of orthophosphate
Acetyl CoA – activated acetate
Water excluded from active site of Ribosome
Glycogen phosphorylase (cleave phosphorylytically, not hydrolytically to keep G1P
in cell)
Hexokinase on binding glucose, water would prevent phosphorylation
Pyrophosphate hydrolysis Drives: RNA synthesis, DNA synthesis, activates amino-acyl to charge tRNA,
citrulline  arginosuccinate (urea cycle), formation of UDP-glucose in glycogen
synthesis, drives activation of FA to acyl CoA before degradation
Urea cycle: Citrulline + asparatate + ATP  AMP + Ppi + argininosuccinate
(argininosuccinate synthetase)
Uses ATP hydrolysis ρ protein
Topoisomerase II
Helicase
Clamp loader
Ubiquitination
P loop NTPase DnaB – Helicase A2 (pg. 797)
DnaA – binds OriC during replication
19S component of proteasome: isopeptidase cleaves off Ub
α subunit of heterotrimeric G protein (P loop participates in nucleotide binding)
small G protein (Ras) – monomeric, cycle between GTP and GDP-bound form. – in
EGF pathway
α3β 3 subunits in hexameric ring of ATP synthase
ENZYME MECHANICS: Conformational change in an enzyme to form the substrate binding site induced by
Induced fit binding of the substrate
adenylate kinase, Hexokinase, Phosphoglycerate kinase, Pyruvate kinase
Citrate Synthase: binds OAA, then acetyl CoA and only hydrolyzes citryl CoA
Approximation The binding of two substrates to a single surface on the enzyme to increase the rate
of their reaction (correct orientation of binding increases reaction)
NMP kinases – pyrimidine metabolism 713
Carbonic anhydrase CO2  bicarbonate
Stabilization of Th intermediate The interaction among enzyme groups and the transition state of a reaction that
lower the activation energy of the reaction and hence increase its rate
Serine proteases: chymotrypsin, subtilisin (Serine’s –OH acts as a nucleophile
Peptidyl transferase center of ribosome (on 23S rRNA)
Rnase P (remove nts from precursor tRNA, and produces correct 5’ termini)
Multi-subunit enzymes α-1,6-glucosidase and transferase on 1 polypeptide chain. (glycogen degradation)
FA synthase: has acetyl Transacylase, malonyl transferase, acyl-malonyl ACP
condensing enzyme (domain 1), (domain 2) 3-hydroxybutyrl ACP dehydratase,
enoyl ACP reductase, β -ketoacyl ACP reductase, ACP, (Domain 3): thioesterase
Bifunctional PFK2 enzyme (controls glycolysis/gluconeogenesis): FBPase2/PFK2
Processivity In DNA synthesis (DNA pol III + β 2 sliding clamp)
RNA synthesis (RNA pol II)
glycogen phosphorylase activity (30Å from catalytic to glycogen binding site)
Primer needed Glycogenin – glycogen synthesis
DNA synthesis – RNA primer
2nd messengers cAMP, calcium, IP3, lipid DAG

Which dehydrogenase can utilize either Glutamate dehydrogenase (amino acid degradation)
NAD+ or NADH+?
Enzymes that move around phosphate Phosphoglycerate mutase (3PG  2,3 BPG  2 PG) – glycolysis
groups Phosphoglucomutase (G1P  glucose 1,6 BP  G6P) – glycogen breakdown
Use PLP Aldehyde group of coenzyme forms a Schiff base with a specific lysine side chain of
the enzyme.
Transaminase uses PLP during amino acid degradation (forms Schiff base)
Glycogen phosphorylase uses PLP to cleave glycogen phosphorylytically
Where is FADH2 made? Succinate dehydrogenase – CAC
Glycerol phosphate dehydrogenase – oxidize glycerol
Fatty acyl CoA dehydrogenase – oxidize fats
Transfer electrons from FADH2 to Q to form ubiquinol (QH2)
Effect of phosphorylation Pyruvate dehydrogenase: inactivated
Glycogen phosphorylase: activated
Glycogen synthase: inactivated
PK: inactivated
HMG-CoA
PFK2/FBase2: turns on PFK2
Use Mg2+ DNA polymerase
Hexokinase, Adenylate kinase, other kinases
Dehydrogenases GAP, alcohol, lactate, isocitrate, α-ketoglutarate, succinate, malate DH, an enzyme
that oxidizes a substrate by transferring one or more protons and a pair of electrons
to an acceptor, usually NAD/NADP