Lecture 8 (Chapter 11)

Lipids: water-insoluble molecules that are highly soluble in organic solvents

Amphipatic: Amphi (both), pathic (suffering/feeling), Having 2 sides with opposite
properties. Having polar and non-polar end.
Philic: (love) so amphiphilic loves both
Five Classes of Lipids:
Fatty Acids(free fatty acids/nonesterified): simplest type of lipid that vary in
hydrocarbon chain length. Main source of fuel and major building blocks of
membranes. Can be saturated or unsaturated.
Triacylglycerol: storage form of fatty acids and major source of glycerol.
Phospholipids: main component of membranes. Consists of fatty acids attached to a
scaffold that bears charged phosphoryl group, creating a macromolecule with a
polar head and nonpolar tail.
Glycolipids: lipids that are bound to carbohydrates. important membrane
constituents
Steroids: polycyclic hydrocarbons with a variety of functions. Function as
hormones that control a variety of physiological functions. Most common is
cholesterol (vital membrane component)

Fatty Acids: amphipathic molecule that possesses both polar and nonpolar
groups.

Carboxyl group is the head that is polar, hydrophilic (pKa ~ 2) is ionized at pH 7.
Tail: nonpolar, hydrophobic

When glucoses (partially oxidized) and fatty acids are broken down, they are
converted into CO2 and H2O. Fats yield more energy than carbohydrates when
undergoing combustion to carbon dioxide and water.

Fatty acids can have saturated or unsaturated acyl chains. Saturated: only single
bonds. Unsaturated: has double bonds in hydrocarbon tail

Nomenclature/Notation for fatty acids A:B, A is the number of carbons and B is
the number of double bonds. C=O bond should not be included when asked for # of
double bonds.

*do not have to remember common names of fatty acids

Carbonyl carbon is carbon #1.

A:B(cis-^1,2,3,4) 1: is the first carbon that a double bond starts at.

Also there is (omega) nomenclature: reversed numbering (last carbon in chain is
first) Fatty acid is named after first double bond that appears (omega)-# fatty
acid. Last carbon in chain is called the omega carbon atom.
Carbon atoms 2 and 3 are often referred to as alpha and beta respectively.

Most of the common fatty acids found in nature have an even number of carbon
atoms and no more than 4 double bonds (typically between 14 and 24 carbons) 16
and 18 are most common.

anoic no double bond
enoic double bond present

Just like amino acids, fatty acids are ionized at physiological pH so preferable to
refer to their carboxylate from vs carboxylic.
Mono vs. poly fatty acids: based on number of double bonds

There are cis and trans unsaturated fatty acids.

Trans fatty acids: trans fat, shape is not changed (partial hydrogenation of fatty
acids, processed) trans double bonds are less prone to oxidation

Cis fatty acids: theres a kink, linear structure/shape is somewhat changed

Most naturally occurring unsaturated fatty acids almost always are the cis
configuration.

Trans/ cis, saturated/unsaturated affects melting temperature.
Unsaturated fatty acids have lower melting points than those of saturated fatty acids
of the same length.
Lower melting T correlates with more fluidity.
Van Der waals interactions occur between hydrocarbon tail in fatty acids More van
der waal interactions present lead to a higher amount of heat which means a higher
melting temp and more energy is needed to separate the van der waal interactions.

The more kinks (cis double bonds) the easier it is to separate van der waal bonds,
and the lower the melting temperature. So cis double bonds lower melting
temperature.

Trans double bonds look like saturated fatty acids so its melting temp is still higher
than cis.

Chain length also affects the melting point. Short chain length and cis unsaturation
enhance the fluidity of fatty acids and lowers melting temp.
Unsaturated, cis fatty acids are known to be good fats because we cannot
synthesize them.
Some fatty acids are essential components of our diet (omega-3 and omega-6)
Concentration of free fatty acids in cells or the blood is low because free fatty acids
are strong acids. This would disrupt the pH balance of cells.
Fatty acids required for energy generation are stored as triacylglycerols(3
fatty acid chains bonded to a glycerol, each carbon of the glycerol is esterified). there
are simple triacylclycerol (same fatty acid chains) and mixed (different fatty acid
chains)

Common soaps are the sodium or potassium salts of fatty acids generated by
treating triacylglycerols with strong bases.
A gram of nearly anhydrous fat stores more than six times as much energy as a gram
of hydrated glycogen.

Most natural plant and animal fat are triacylglycerols
Glycogen and glucose stores can maintain biological activity for about 18-24 hours.
Tg stored allows survival for several weeks

Triacylclycerol is a good fuel:
1) fatty acids are richer in energy (more reduced) than carbohydrates.
Complete oxidation of Tg yields 38 kJ energy/ig (more than 17kJ/g of protein
or carbohydrate)
2) tg can be stored more efficiently. In a neutral way that does not affect cellular
processes
3) tg aggregates are inert and there is no risk of undesired chemical reactions
with other cellular components
4) provides enough stored energy to last weeks

Soap is made up of sodium and potassium salts of long chain fatty acids

Soap is an emulsifying agent. Aqueous liquid with organic solvent and
hyrophobic/hydrophilic parts.

Adipose cells are specialized for the synthesis and storage of triacylglyceros and for
their mobilization into fuel molecules that are transported to other tissues by the
blood. Adipose tissue also serves as a thermal insulator.

3 common types of membrane lipids:
1) Phospholipids: has to have a phosphate group
2) Glycolipids: has to have glucose
3) Cholesterol: does not contain fatty acids

Lipids: can be fatty acids or isoprenoids/steroids (cholesterol)

Fatty acids phosphoglyerides/glycerophospholipids,
trigylcerides/triacylglycerides. Sphingolipids

Phospholipids are abundant in all membranes. Composed of one or more fatty acid
attached to a platform attached to a phosphate which is in turn attached to an
alcohol. (Backbone is usually a sphingosine or glycerol)

Phospholipids come from glycerides and sphingolipids. They have hydrophilic and
hydrophobic components.

Phosphoglyceride has a glycerol as a backbone. Only first 2 carbons of
phosphorglyceride are esterified to fatty acid chains. 3 carbon is bonded to
phosphate and alcohol. Simplest phosphoglyceride is phosphatidate.

Phosphotidates: only small amounts present in membranes. But is a key
intermediate in the biosynthesis of other phosphoglycerides and triacylglycerols.
Made up of Phosphotidyl- ethanolamines, serines, cholines, inositols and
cardiolipin are the alcohols that can be found bonded to phosphate.

Sphingolipids: have a sphingosine backbone instead of glycerol. Have a
hydrophobic tail. (sphingomyelin is common)
Sphingosine is a amino alcohol that contains a long unsaturated hydrocarbon chain.

Sphingomyelins: phospholipids found in membranes. Sphingosine backbone. It can
be phosphoryl(choline/ethanolamine). Fatty acid attached via an amide linkage
to the sphingosine backbone
Sphingomyelinphospholipids

Glycolipids are sugar-containing lipids. Derived from sphingosine. Differ from
sphingomyelin because it is linked to one or more sugars instead of
phosphorylcholine.
Simplest glycolipid is a cerebroside. More complex are gangliosides.

Cerebrosides/gangliosides glycosphingolipids (always have a sphingosine
backbone attached at the 1-OH group of sphingosine)

One sugar: cerebroside
Two or more sugars: ganglioside

In ceramide, fatty acid is attached via an amide to sphingosine backbone
Ceramides sphingomyelin, cerebrosides, gangliosides

Steroids: lipids that have a variety of roles. Made up of cyclohexane rings and
cyclopentane. Powerful hormones, facilitate the digestion of lipids in the diet, are
key membrane constituents.

Steroid nucleus: made up of 3 cyclohexaness and 1 cyclopentane

Cholesterol: made of isoprene units. Most common steroid. Important in
maintaining membrane fluidity. Absent from prokaryotes but found in all animal
membranes. Constitutes almost 25% membrane lipids but absent from some
intracellular membranes. Free cholesterol does not exist outside of membranes
(esterified to fatty acid). Cholesterol disrupts the tight packing of fatty acid chains

Cholesterol derivatives:
1) 5 families of steroid hormones: androgens, estrogens, progestins,
glucocorticoids, mineralocorticoids
2) bile acids: assist in absorption of dietary lipids in the intestine
3) vitamin D ( calcium absorption)


Membrane lipids are amphipathic (hydrophilic and hydrophobic)

3 types of lipid anchors (proteins covalently bond to lipids to localize the protein
to the cell membrane)

1) palmitoyl group attached to a cysteine residue by a thioester bond
2) Farnesyl group attached to a cysteine residue at the carboxyl terminus
3) Glycosylphosphatidylinotisol (GPI) anchor: glycolipid structure
attached to the carboxyl terminus

GPI anchor is always found on the outside of the cells

Hutchinson-Gilford Progeria Syndrome (HGPS): caused by inappropriate
fanesylation. Mutation in the gene for the protein lamin.
Lamin processing is different in HGPS patients (progerin instead of lamin A)

Eukaryotic membranes: membranes serve as boundaries that maintain division of
labor in cell. Membranes are actively involved in cellular processes. Permeability
barriers

Membranes are bilayer. Phospholipis and glycolipids form lipid bilayer in aqueous
solutions. Polar head groups + hydrophobic tails

Membranes formation: lipids form ordered structures spontaneously in water
Driving force behind amphipathic lipids to form ordered structures in aqueous
solutions is: waters tendency to form H-bonds and share in polar interactions.
Hyrophobic affect promotes self association of lipids in water to maximize water
entropy

Membrane composition reflects its function: membrane fluidity is controlled by
fatty acid composition and cholesterol content.



Biological membranes are asymmetric and heterogenous. Made up of outer and
inner leaflet.
The asymmetry of membrane structure is functionally important to the membrane.


Lecture 9: Chapter 12 Membranes

Membrane fluidity controlled by fatty acid composition and cholesterol content.
Membrane proteins diffuse laterally in the membrane
Proteins carry out most membrane processes
Membrane proteins are transporters.

The thickness of most membranes is between 60 and 100 Angstroms (6 and 10 nm).
Consist mainly of lipids and proteins, as well as carbohydrates linked to the lipids
and proteins.
Membranes are noncovalent assemblies, assymetric, and fluid structures. Most cell
membranes are electrically polarized such that the inside is negative.

Vanderwaals forces: hydrocarbon tails
Electrostatic and hydrogen bonding: polar head groups and water molecules

Lipid vesicles or liposomes can be formed from phospholipids.

This transition temperature depends on the length of the fatty acid chains and on
their degree of unsaturation. Long fatty acids interact more strongly because of the
increased number of van der Waals interactions than do short ones and thus favor
the rigid state.

The presence of saturated fatty acid resi-dues further favors the rigid state because
their straight hydrocarbon chains interact very favorably with one another.

On the other hand, a cis double bond produces a bend in the hydrocarbon chain.
This bend interferes with a highly ordered packing of fatty acid chains, and so Tm is
lowered.

Cholesterol helps maintain membrane fluidity.

Membranes are mad of 2 layers of phospholipids, hydrophobic tail and hydrophilic
head. Form a bilayer membrane.

Melting temperature of fatty acids is affected by length, # of double bonds, and
cis/trans configuration. So membranes have different melting temperature.

Below melting temperature, membrane is rigid, solid (ordered structure) because of
van der waal interactions. Above melting temperature, membrane gains fluidity.

In animals, membranes have cholesterol. Cholesterol disrupts the van der waals
interactions in the tight packing of the fatty acid chains, which makes the membrane
more fluid. At higher temperatures, cholesterol is very rigid which limits ability of
chains to move around. Limits the range of fluidity/solidity of membranes.

Membranes are 2-dimensional solutions of oriented globular proteins and lipids.



Properties and characteristics:
1) Sheet like structures
2) Composed of lipids and proteins
3) Membrane lipids are small amphipathic molecules
4) Proteins serve to mitigate the impermeability of membranes
5) Membranes are noncovalent assemblies. Hydrophobic affect
6) Membranes are asymmetric
7) Membranes are fluid structures

Layer that faces cytoplasmic side: inner leaflet
Layer that faces extracellular space: outer leaflet. Anything with sugars are on the
outer leaflet (glycolipids)

Transverse Asymmetry: between leaflets.
Lateral Heterogeneity: (along membrane) Different regions have different
concentrations of lipids/proteins. Not evenly distributed.

Certain enzymes (called flipaes) flip lipids back to where they belong.

Lateral diffusion: lipids diffuse through the membranes very rapidly (2uM/sec)
Transverse diffusion (flip-flopping) is very slow

The fluid mosaic Model:
Cholesterol, oligosaccharide side chain(OUT), peripheral protein(IN), integral
proteins(IN), phospholipid membrane, glycolipid(OUT).

Membranes composed almost entirely of lipids are suitable for insulation because
hydrophobic components do not conduct currents well. The plasma or exterior
membranes conduct the traffic of molecules into and out of the cells. Protein content
of plasma membranes is around 50%. Energy transduction membranes
(mitochondria/chloroplast) contain higher amount of protein, around 75%.

Heterokaryons: consists of 2 different cells (human and mouse cells) Then the cells
are fused (proteins on the cell surface are detected by specific antibodies), it then
becomes one big heterokaryon and after some time (40 min) the human and mouse
proteins are intermixed in the plasma membrane.

Fluorescence Recovery after photobleaching (FRAP):
Starts with one cell then a laser beam is pointed at specific area in cell which
bleaches the fluorescence. After bleaching certain region, green fluorescence protein
will denature (unfold) so the protein is no longer able to fluoresce. See how long it
takes to recover/ gain fluorescence. GRAPH on slide 11. Some proteins move faster
than others so you can observe this with FRAP. USES lateral diffusion.

Different proteins associated with membrane processes:
Transporters
Anchors
Receptors
Enzymes


Membrane Proteins are classified as Integral or Peripheral:

Integral membrane proteins enter hydrophobic environments (can enter both
leaflets or just one) can be released only when physically disrupted.
Peripheral proteins: interact with the outside of the head (hydrophilic). They can be
bound to the integral proteins. Primarily bound to the head groups of lipids by
electrostatic and hydrogen-bond interactions.

Differentiating is based on how easy it is to remove protein from membrane
(peripheral does not need treatment, easier) Integral protein needs full dissocation
(harder to remove.)

Proteins in membranes are associated with alpha helices and beta
strands/pleated sheets. Portions that are in contact with nonpolar core of the
lipid bilayers are dominated by alpha helices or beta sheets because these second
order structures neutralize the polarity of the peptide backbone through h-bond
formation.

Integral membrane proteins can embed part of the protein onto the membrane.
Dimers form hydrophobic channel (hydrophobic amino acid side chains)
Prostaglandin H2 synthase-1 (involved in pain from injury)
The channels formed create a hydrophobic environment. Serine 530 (Ser 530) is a
region within hydrophobic channel that aspirin can modify and inhibit
cyclooxygenase activity by obstructing the channel. So aspirin reduces
prostaglandin and pain.

Membrane-spanning alpha-helices are a common structural feature of integral
membrane proteins.
Also beta strands can be used to form a pore in the membrane.

Aspirin inhibits cyclooxygenase activity by obstructing the channel.

Single transmembrane segment: e.g. glycophorin A is an integral protein in the
membranes of red blood cells.

Hydropathy plots: x axis is residue number, y axis: hydropathy index. Hydrophilic:
have negative numbers. Hydrophobic have positive numbers (single
transmembrane protein).

Transport across membranes:
Simple diffusion
Facilitated diffusion (passive transport)
Simple and facilitated both have transport from high to low concentrations
(thermodynamically favored direction, no energy input required) e.g pores,
channels, carriers.

Active transport: moves in thermodynamic unflavorable direction (low to high
concentrations) needs energy to drive the process. E.g. pumps

[C2]-[C1] is the concentration gradient.
High concentration has lower entropy, more ordered.
Low concentration has higher entropy
Molecules spontaneously move from a region of higher concentration to one of
lower concentration.

Lipophilic molecules can pass through the membrane because the dissolve in the
lipid bilayer.
Na+ cannot enter because it is a charged ion.

In charged molecules, charge differences across the membrane must be taken into
account.

A nerve impulse, or action potential, is an electrical signal produced by the flow of
ions across the plasma membrane of a neuron. In par-ticular, Na+ transiently flows
into the cell and K+ flows out.

Inside of membrane cells are high in potassium concentration (lower on the
outside). Because of the difference in charge, potassium cannot be balanced equally.


Extracellular: positively charged
Intracellular: negative

Simple diffusion: movement until concentration inside and outside is the same.
Thermodynamically favorable

Lipid bilayet (nonpolar,hydrophobic) are more impermeable(less permeable) to
ions and most polar molecules. Based on a log scale.

Ability of small molecules to cross a membrane is a function of its hydrophobicity.

Facilitated diffusion using carriers (from high to low concentrations) does not
require energy. Requires a carrier protein. Transmembrane protein carriers
changes shape to facilitate entry and exit of some nutrients.

Facilitated passive diffusion (uses pores/channels) this is regulated because it can
be closed or open. 1) ligand activated, 2) voltage activated. Channels display a
measurable affinity and specificity for the transported solute. Channels are gated

Ligand activated channels: ligand is a signaling molecule. Ions can go in when gate
opens. When ligand dissociates, gate closes again.

Voltage activated channels: action potential: at rest, negative charges are inside.
Once event triggers signaling, action potential enters and negative charges change to
positive (change in voltage opens channels and sodium flows in). Potassium
channels also open and allow K+ to flow out of cell.

Channels have affinity filters that only allow specific ions to flow in or out.

Pottasium channel: higher K+ concentration has larger diameter (10 Angstroms)
where K and Na can go in and lower K+ concentration has smaller diameter (3
angstroms) where only K+ can go in. Hydration shell needs to be lost to allow K+
ions to enter the narrow part of channel.

Thermodynamically unfavorable to lose hydration shells is made up by the carbonyl
oxygens from selective filters.

Selective filters are carbonyl oxygens. Interactions can only take place with 2
carbonyls at a time. (slide 40)

K+ channel transport continued: K+ repels which pushes a K+ out of the cell.

Facilitated vs Simple Diffusion:

Both transport molecules down their concentration gradient.
Simple diffusion has linear form between [S] and velocity.
Facilitated diffusion has a curved first increasing then slope decreases. (curved) this
is because of the saturation of carriers (when carriers are gone, process is slower)

Active transport: unfavorable. Requires energy. Energy can come from ATP
hydrolysis, light energy, and energy stored in ion gradients. The process is either
electrogenic or electroneutral.

ATP: uses a Na+ -K+ ATPase pump. 3 Na+ enters and 2 K+ leaves. ions used.
Maintenance of the gradient consumes a large amount of energy (20%-40% of total
metabolic energy) Releases ADP.

3 Na+ goes in, atp phospholates are hydrolyzed and changes the shape of protein.
The shape change releases Na+ and enables K+ to bind to the pump. Release of Pi
returns the pump to its original shape releasing K+ to the cells interior and exposes
Na+ binding sites. Cycle repeats.

Secondary transport system: transport of molecule uses ion gradient in different
organisms or tissues. Can be either antiporters or symporters.

Na+ glucose symporter.

Lecture 10- Chapter 6 Basic Concepts of Enzymes


Enzymes accelerate reactions by factors of as much as a million or more.
Carbonic anhydrase is one of the fastest enzymes known.

Enzymes are highly specific both in the reactions that they catalyze and in the choice
of reactants, called substrates.

Proteolytic enzymes (in vivo catalyze proteolysis, the hydrolysis of a peptide
bond)

The specificity of an enzyme is due to the precise interaction of the substrate with
the enzyme. Precision is a result of the intricate 3-d structure of enzyme protein.

6 Major Classes of Enzymes:
1) Oxidoreductase: transfer electrons between molecules. Catalyze oxidation-
reduction reactions.
2) Transferases: transfer functional groups between molecules.
Aminotransferases are prominent in amino acid synthesis.
3) Hydrolyases: cleaves molecules by addition of water. Trypsin is an example.
4) Lyases: adds atoms or functional groups to a double bond or removes them
to form double bonds. Lyase fumarase is crucial to fuel metabolism
5) Isomerases: move functional groups within a molecule.
6) Ligases: join 2 molecules at the expense of ATP hydrolysis.

Catalytic activity of many enzymes depends on the presence of cofactors.
Apoenzyme: enzyme without its cofactor
Holoenzyme: complete, catalytically active enzyme.

Cofactors: 1) small organic molecules (derived from vitamins called coenzymes)
2) metals

Prosthetic (helper) groups: tightly bound coenzymes
Coenzymes differ from substrates because the are used by a variety of enzymes.
Different enzymes that use the same coenzyme usually carry out similar chemical
transformations.

Free energy (G): thermodynamic property that is a measure of useful energy, or
energy capable of doing work.

The difference in energy between products and reactants determines whether a
reaction will take place spontaneously.
Free energy required to initiate the conversion of reactants into products
determines the rate of reaction.

A reaction can take place if G is negative. spontaneously or without input of
energy. Also called exergonic reaction

An input of free energy is required when G is positive. (not spontaneous) also called
an endergonic reaction.

A system at equilibrium means G is zero and there is no net change in concentrations
of products and reactants.
The G is independent of path or molecular mechanism

G provides no information about rate of reaction.

The standard state is defined as having pH = 7. So when H+ is a reactant, its
concentration is 1 M. (concentration of water is also 1 in this state)

An enzyme cannot alter the laws of thermodynamics and cannot alter the equilibrium of a
chemical reaction.

Enzymes accelerate the attainment of equilibria but do not shift the position. The
equilibrium position is a fxn of only free-energy difference between reactants and
products.

Transition state: (double dagger) fleeting molecular structure that is no longer the
substrate but not yet the product. Least stable and most-seldom occurring species along
reaction pathway because it has the highest free energy.

Free energy of activation or Activation energy is the difference between transition
state and substrate.
Enzymes facilitate the formation of the transition state. Catalysis is based on stabilization
of transition state.

Enzymes bring together substrates in enzyme-substrate (ES) complexes. The substrate
or substrates are bound to specific region of the enzyme called the active site.

The interaction of the enzyme and substrate at the active site promotes the formation of
the transition state. Active site is the region of the enzyme that most directly lowers the
G (transition state/double dagger)

Common features of active sites:
Active site is a 3-d cleft or crevice formed by groups that come from different parts of
amino acid sequence.

Active site takes up a small part of the total volume of enzyme. Most of the amino acid
residues in an enzyme are not in contact with the substrate. Extra amino acids serve as a
scaffold to create 3-d structure and constitute regulatory sites (sites of interaction with
other proteins or channels to bring the substrates to the active sites).

Active sites are unique microenvironments: water is usually excluded from active site
unless it is a reactant. Nonpolar microenvironment enhances the binding of substrates as
well as catalysis.

Substrates are bound to enzymes by multiple weak attractions: noncovalent bonds
mediated by the hydrophobic effect (van der waals, electrostatic, and hydrogen bonds).
To bind as strongly as possible, enzyme and substrate should have complementary
structures.

Specificity of binding depends on the precisely defined arrangement of atoms in an active
site. (need matching shapes to fit together)

Lock and key analogy. Induced fit: active site assumes shape that is complementary to
that of the substrate only after the substrate has been bound.

Binding energy: free energy released from the binding of substrate and enzyme.
Full complement of interactions between enzyme and substrate is formed only when the
substrate is in the transition state. (Maximal binding energy is at transition state).

Transition-state analogy: compounds that resemble the transition state of a reaction but
are not capable of being acted on by the enzyme.
The inhibitor power of transition state underscores the essence of catalysis: selective
binding of the transition state.

Catalytic antibodies or abzymes: antibodies generated that recognize the transition
states of certain reactions.

Lock and key model is harder to bend (tightly bound)

Active site is complementary to transition state.
Enzyme stabilizes the transition state.
Enzyme inhibitors work best when it looks like a transition state, not a substrate.
Proline racemase L-Proline transition state D-Proline
Transition-state analog (pyrrole 2-carboxylic acid)

Enzyme Kinetics Lecture 11 (Chapter 7)
Allosteric enzymes: prevent chaos and allows for the efficient integration of
metabolism.

Kinetics is the study of rates of chemical reactions.

2 Types of Enzymes:
Michaelis-Menten: their activity is a function of substrate concentration
Allosteric: regulated, information sensors

Enzymes exert kinetic control over thermodynamic pontentiality (forward or
backward)

Kinetics: First Order Reactions: velocity is directly proportional to reactant
concentrations (units s-1)
S P
V = -d[S]/dt = d[P]/dt

Two reactants second-order reactions or bimolecular reactions

2A P
or
A + B P

V = k[A]
2
or V = k[A][B]

Some cases have zero order reactions which means rate is independent of
concentrations.

Irreversible:
V = k[S] (V = velocity) depends on concentration of substrate and rate constant k


Why does the first order graph (product vs time) eventually level off? running out
of substrate (analogy to popcorn)

Linear relationship between reactant concentration [S] and initial velocity Vo. Slope
= k (time-1)

Reversible reactions: At equilibrium k1[S] = k-1[P]

Biomolecular reactions where S2 is in excess of S1, reaction becomes pseudo first
order. S1 + S2 P1 + P2 (graph for product vs time looks like first order reaction)
Linear curve between V vs [S1] in pseudo first-order. [S2] >> [S1]

Effect of enzyme concentration on initial velocity at a fixed saturating substrate
concentration ([S]>>>[E])
Pseudo first-order reaction

Kinetic of Enzyme catalyzed reactions (keeping [Enzyme] constant but increasing
[substrate]Initially first-order reaction with respect to S but levels off to zero
order reaction with respect to S (Vo vs [S])

Zero order reaction: adding more substrate no longer affects velocity because of
saturation. (Every enzyme has gone to substrate) Vo reaches V max.

E + S ES E + P (all reversible)
K1/K-1, K2/K-2
K = rate constant

Enzymes are neither reactants nor products


Michealis-Menten (MM) equation: describes variation of Enzyme Activity as a
function of Substrate Concentration

E + S ES E + P
When ES complex is formed, it can either dissociate to E + S (k-1)or proceed to form
product (k2)

MM Equation: V0 = Vmax[S]/ Km + [S]

V0 = initial velocity
Vmax = Maximal velocity
Km = Michaelis Constant is unique to each enzyme and independent of enzyme
concentration.
Km = (K2 + K-1)/K1
[S] = substrate concentration

Affinity of E for S is inversely proportional to Km

Vmax = K2[Et] (directly dependent on enzyme concentration)

V0 = Vmax[S]/(Km + [S])
When [S] is much less than Km Vo = (Vmax/Km)[S]
When [S] is very large Vo = Vmax
[S] = Km when Vo = Vmax/2
V0 = Vmax[S]/2[S]
To reach Vmax, need infinite amount of substrate concentration [S]

Physiological consequences of Km
Mitochondrial aldehyde dehydrogenase has low km but inactive in certain
population. (having more of this allows you to control the toxic effects of drinking
too much)
Cytoplasmic aldehyde dehydrogenase has high Km.
Low Km mitochondrial form vs high km cytoplasmic form.
High km high rate of catalysis only at very high concentrations of acetaldehyde
less converted to acetate and excess acetaldehyde escapes into the blood.

Evidence suggests that the Km value is approximately the substrate
concentration of the enzyme in vivo.

A double reciprocal the Lineweaver-Burk plot
Y = a x +b
1/Vo = Km/Vmax * 1/S + 1/Vmax
slope: Km/Vmax
Y axis: 1/Vo
X-axis: 1/[S]

For most enzymes: Km lies between 10^-1 and 10^-7 (depends on pH, temperature,
ionic strength)

Turnover Number = number of substrate molecules that an enzyme can convert
into product per unit time when the enzyme is fully saturated with substrate.
Turnover number = k2 or Catalytic constant (kcat) Most turnover numbers are
between 1 to 10^4 per second

When the enzyme is saturated with substrate K2 = Kcat

Kcat = Vmax/[Et]
Measures the effectiveness of an ezyme. Usually the limiting step.
Catalytic Efficiency: if [S] << Km, we can assume that free enzyme [E] = [Et] so Vo =
(kcat/kM)[S][ET]

Kcat/Km = catalytic efficiency

Most reactions in biological systems are Bisubstrate Reactions:
A + B P + Q (reversible)

Sequential reactions: random vs ordered: all substrates must bind to the enzyme
before any product is released. A ternary complex consisting of the enzyme and both
substrates forms.

Conversion of pyruvate to lactate by lactate dehydrogenase

Double displacement/ping-pong reactions: one or more products are released
before all substrates bind the enzyme. Existence of a substituted enzyme
intermediate in which the enzyme is temporarily modified.



Allosteric enzymes are catalysts and information sensors. Allosteric enzymes
control the flux of biochemical reactions in metabolic pathways.

Most enzymes in the cell are Michaelis-Menten enzymes.

Catalysis is not enough in functioning of a cell. Vast array of reaction pathways also
need to be regulated.
Allosteric enzymes: regulate the flux of biochemical through metabolic pathways.

Key features of allosteric enzymes: regulation of catalytic activity by
environmental signals, including the final product of the metabolic pathway
regulated by the enzyme. Kinetics for allosteric enzymes are more complex than M-
M and they have quartenary structures with multiple active sites.

Feedback inhibition: common means of biochemical regulation. Bears no
structural resemblance to the substrate or product of the enzyme that they inhibit.
Feedback inhibitors do not bind at the active site but rather at a distinct regulatory
site on allosteric enzyme. (allos : other, stereos: structure)

Allosteric enzymes always catalyze the committed step of metabolic pathways.
They can recognize inhibitor molecules and stimulatory molecules.

Allosteric enzymes differ from M-M. Graph is sigmoidal with sharp increase of o
in the middle of the curve. More sensitive to changes in [S] near Km which allows for
more sensitive control of reaction velocity.

Allosteric enzymes are quaternary structures.
Concerted/MWC model: have multiple active sites on different polypeptide chains.
Can exist in R (relaxed) or T (tense) state. R is active conformation which catalyzes
reactions. T is less active. In the absence of substrate or signal molecules, R and T
are in equilibrium. T/R is the allosteric constant Lo. Concerted model requires that
all of the subunits or active sites must be in the same state (all R or all T) This is
called the symmetry rule. S binds more readily to the R form than to the T form.

The binding of substrate disrupts the T R (reversible) equilibrium in favor of R.
This is called cooperativity and accounts for the sharp increase in Vo.
Cooperativity means that allosteric enzymes display a threshold effect. Below
certain [S], there is little to no enzyme activity. After threshold is reached, enzyme
activity increases rapidly.

Signal Sensing capabilities: Positive effector binds to the R at a regulatory site and
stabilizes this form (R to S more likely). Negative effector binds to T and stabilizes it
(making R S binding less likely). Positive effector lowers the threshold
concentration of substrate needed for activity. Negative effector raises the
threshold.

Heterotropic effectors (regulatory molecules affect allosteric enzymes): activators:
shift sigmoidal cure to the left. Inhibitors shift it to the right. ATP is an activator. CTP
is an inhibitor.

Homotropic effects (substrates affect allosteric enzymes): account for the
sigmoidal nature of the kinetics curve.

Sequential model: binding of substrate at one site influences the substrate binding
to neighboring sites without necessarily inducing a transition encompassing entire
enzyme. (negative cooperativity: binding of one substrate decreases the affinity of
other sites for the substrate)

Ensemble studies: experiments on enzymes.

Enzyme Mechanism and Inhibition Lecture 12 Chapter 8

Covalent catalysis: active site contains a reactive group, usually a powerful nucleophile
that becomes temporarily covalently modified in the course of catalysis. (proteolytic
enzyme chymotrypsin)

General Acid-Base Catalysis: a molecule other than water plays the role of proton
donor/acceptor. Chymotrypsin uses histidine residues as a base catalyst to enhance the
nucleophilic power of serine.

Metal Ion Catalysis: metal ions can function catalytically in several ways. Either
serve as a electrophilic catalyst (stabilizing negative charge on reaction
intermediate). Metal ion may generate a nucleophile by increasing acidity of nearby
molecule. Or may bind to substrate, increasing the number of interactions with the
enzyme and thus the binding energy.
Catalysis by approximation and orientation: reaction rate may be considerable
enhanced by bringing the 2 substrates into proximity.

Temperature: as temperature rises, rate of most reactions increase (more
Brownian motion) For enzyme activity: there is a temperature at which the increase
in catalytic activity ceases and there is a precipitous loss of activity. When T gets too
high, weak bonds are not strong enough to withstand the polypeptide chains
thermal jostling and protein loses structure (denatured).
Endotherms (humans): effect of outside temperature on enzymes is minimized.
Exotherms: temperature is an important regulator of biochemical an biological
activity.


PH: Maximal pH of enzymes varies and is correlated with the environment of the
enzyme. Pepsin is very effective in pH 1-2. Chymotrypsin is effective in pH 8.

Irreversible inhibitors: dissociates very slowly from its target enzyme because it
has been tightly bound to the enzyme (covalently or noncovalently).

4 groups: group-specific reagents, affinity labels (substrate analogs), suicide
inhibitors, and transition-state analogs.

Group-specific reagents: DIPF Chymotrypsin, serine

Affinity labels: molecules that covalently modify active-site residues and are
structurally similar to an enzymes substrate. (TPCK) nad chymotrypsin

Suicide inhibitors (mechanism-based inhibitors) are chemically modified
substrates. Provide researches with the most-specific means of modifying an
enzymes active site. Inhibitor binds to enzyme as a substrate and is initially
processed by the normal catalytic mechanism. Creates a chemically reactive
intermediate that inactivates the enzyme through covalent modification. Enzyme
participates in its own irreversible inhibition suggests that the covalently modified
group on the enzyme is catalytically vital.

Transition state analogs: potent inhibitors of enzymes. Inhibitory power supports
the role of transition-state formation in catalysis.

Penicillin: inhibits bacterial growth by mimicking the D-Ala-D-Ala moiety. Acts a
suicide inhibitor

Proteolytic enzymes of proteases: cleave proteins by a hydrolysis reaction.
Chymotrypsin.

Chymotrypsin is a good example of the use of covalent modification as a catalytic
strategy. Uses serine 195. Chromogenic substrate: N-acetyl-L-phenylalanine p-
nitrophenyl ester. Products: p-notrophenolate. The cleavage obeys Michaelis-
Menten. Has 2 steps (first step faster than second). Forms acyl-enzyme
intermediate. (hydrolyzed to form carboxylic acid and regenerates free enzyme)
One molecule of p-nitrophenolate is produced rapidly from each enzyme molecule
as acyl-enzyme intermediate is formed. It takes longer for the enzyme to reset by
hydrolysis of intermediate (both steps are required for turnover) Ping-pong
reaction (double-displacement)

Histidine-57: affinity labeling. Chymotrypsin reacts with molecule that specifically
binds to the active site because it resembles a substrate and then forms a stable
covalent bond with a group on the enzyme that is in proximity. TPCK binds to
chymotrypsin because of its phenylalanine side. Reactive group in TPCK is the
chloromethyl ketone covalently modifies of the ring nitrogens of histidine 57.

Chymotrypsin activity requires serine 195, histidine 57 and aspartic
acid/aspartate 102 (Catalytic triad).

Side chain of serine 195 is hydrogen bonded to imidazole ring of histidine 57. The
NH group of imidazole ring is hydrogen bonded to the carboxylate group of
aspartate 102.
Histidine acts a general base catalyst. Serves to position the serine side chain and
to polarize its hydroxyl group so it is prepared for deprotonation. It accepts the
proton from serine-195 hydroxyl group.
Mechanism*

Tetrahedral intermediate: unstable, bears a negative charge on the oxygen atom
Oxyanion hole: helps stabilize the tetrahedral intermediate by interactions with NH
groups.
Acylation of the enzyme: first stage of the hydrolytic reaction

Deacylation: water molecule takes place of earlier occupied amine component of
substrate. Ester group o the acyl-enzyme is then hydrolyzed . Histidine acts a base
catalyst and draws proton away from water moleculetetrahedral
intermediatecarboxylic acid product release of carboxylic acid readies enzyme
for another round of catalysis.

Binding of an appropriate side chain into this pocket positions the adjacent peptide
bond into the active site for cleavage. Specificity of chymotrypsin depends almost
entirely on which amino acid is directly on amino-terminal side.

No reversible inhibition on exam

















































Hemoglobin Lecture 13- Chapter 9

Hemoglobin is an allosteric protein. It is a component of red blood cells. Carries
oxygen from the lungs to the tissues and contributes to the transport of carbon
dioxide and hydrogen ions back to the lungs.

Both hemoglobin and myoglobin are oxygen binding proteins. Must be able to bind
and release oxygen

Myglobin is in the muscles, storage form of oxygen. Facilitates diffusion of the
oxygen to cellular sites that require oxygen and provide a reserve supply of oxygen
in times of need.

Hemoglobin behaves like allosteric. It displays cooperative behavior (sigmoidal
graph) Myoglobin behaves like M-M (hyperbolic curve).

Allosteric has to have more than one active site and be larger than primary
structure.
Myoglobin: single polypeptide chain consisting of mostly alpha-helices that are in a
globular structure. Globin fold. Can be oxy or deoxymyoglobin.
Myoglobin is a monomeric protein. Hemoglobin is tetrameric. Active site is on
the heme. Myoglobin only has 1 heme (oxygen binding site)

Heme: a prosthetic group that determines whether myo/hemoglobin is able to bind
to oxygen.

The heme group gives muscle and blood their distinctive red color. It consists of an
organic component and a central iron atom. The organic component, called
protoporphyrin, is made up of four pyrrole rings linked by methine bridges to form
a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propio-nate side
chains are attached. Under normal conditions, the iron is in the ferrous F2+
oxidation state. Binding sites at fifth and sixth coordination sites.

Hemoglobin: HbA (adult)

Heme: Fe-protoporphyrin IX has Fe and pyrrole rings surrounding it.

Heme plane is attached to proximal histidine (5
th
site)(imidazole ring of a histidine
residue of the protein) for hemoglobin and myoglobin and distal histidine on
opposite side. In deoxyhemoglobin/deoxymyoglobin 6
th
site is unoccupied
(available for binding oxygen).

Hemoglobin in the lungs has saturation level of 98% and 32% in tissues.
Hemoglobin delivers more O2 to tissues than would myoglobin.

fMRI takes place when iron ion moves into the plane of the porphyrin ir paralleled
by changes in the magnetic properties of hemoglobin.


When hemoglobin is not bound to oxygen deoxyhemoglobin. When it is bound to
oxygen oxyhemoglobin

Hemoglobin made up 2 alpha polypeptide subunits and 2 beta polypeptide subunits.
Deoxyhemoglobin corresponds to the T state of hemoglobin, whereas
oxyhemoglobin corresponds to the R state. In regard to hemoglobin, the T state has
a lower affinity for oxygen than does the R state.

The a1b1 and a2b2 dimers rotate approximately 15 degrees with respect to one
another. The re-arrangement of the dimer interface provides a pathway for
communication between subunits: the presence of oxygen on one of the subunits is
immediately communicated to the others so that the subunits change from T to R in
concert. (allosteric)

Hemoglobin with three sites occupied by oxygen is almost always in the quaternary
structure associated with the R state. The remaining open binding site has an
affinity for oxygen more than 20- fold greater than that of fully deoxygenated
hemoglobin binding its first oxygen atom. However, the behavior is not fully
concerted, because hemoglobin with oxygen bound to only one of four sites remains
primarily in the T- state quaternary structure. Yet, this molecule binds oxygen three
times as strongly as does fully deoxygenated hemoglobin, an observation consistent
only with a sequential model.

Hemoglobin can be extracted from red blood cells and fully purified. Interestingly,
the oxygen affinity of purified hemoglobin is much greater than that of hemoglobin
within red blood cells. The affinity is so great that only 8% of the oxygen would be
released to the tissues if hemoglobin in the cell behaved in the way that purified
hemoglobin behaves. This dramatic difference is due to the presence, within these
cells, of an allosteric regulator molecule 2,3- bisphosphoglycerate ( 2,3- BPG;
also known as 2,3- diphosphoglycerate or 2,3- DPG). This highly anionic
compound is present in red blood cells at approximately the same concentration as
hemoglobin (, 2 mM). 2,3- BPG binds to hemoglobin, reducing its oxygen affinity so
that 66% of the oxygen is released instead of a meager 8%


Conformational changes in hemoglobin alpha1,beta1 alpha2,beta2 interface rotate.

Deoxyhemoglobin (15 degree rotation) oxyhemoglobin

Bigger hole in the middle of deoxyhemoglobin vs oxyhemoglobin

Homotropic effect: disruption of the TR equilibrium by substrates. The
heterotrophic effect: disruption of the TR equilibrium by effectors

Allosteric Effector Molecules that bind Hb: H+,CO2, Cl-, metabolite, 2,3-
biphopshoglycerate (BPG)
All of these bind dexoxyHB better than oxy-Hb, thus promoting release of O2 from
Hb

Fetal Hemoglobin: (HbF): must bind oxygen when the mothers hemoglobin is
releasing oxgen.

No sickle cell anemia on exam.

Hydrogen Ions and Carbon Dioxide promote the release of oxygenThe Bohr effect
More CO2, pH goes down

The regulation of -xygen binding by hydrogen ions and carbon dioxide is called the
Bohr effect after Christian Bohr, who described this phenomenon in 1904.

The oxygen affinity of hemoglobin decreases as pH decreases from the value of 7.4
found in the lungs, at 100 torr of oxygen partial pressure ( Figure 9.18), to pH 7.2
and an oxygen partial pressure of 20 torr found at active muscle.

The heterotropic regulation of hemoglobin by hydrogen ions and carbon dioxide
further increases the oxygen- transporting efficiency of this magnificent allosteric
protein.
Carbonic anhydrase is an enzyme abundant in red blood cells.