UNIT I:

Protein Structure and Function

Enzymes
Overview

• Virtually all reactions in body mediated by

enzymes, which are protein catalysts that
increase rate of reactions without being changed
• Enzymes direct all metabolic events
Nomenclature

• Each enzyme has two names:

– Short, recommended name, convenient for use
– More complete, systematic name used when enz
must be identified without ambiguity
A. Recommended name
– Most commonly used enz’s end with “-ase” attached
to substrate (e.g., glucosidase, sucrase, urease), or to
description of action performed (e.g., lactate
dehydrogenase, adenylyl cyclase)
– Some enz’s retain original trivial names e.g., trypsin,
pepsin
B. Systematic name
- The international union of biochem & mol biol
(IUBMB) set a system in which enzymes are
divided into 6 major classes, each with
numerous subgroups
- Suffix –ase is attached to description of chemical
reaction catalyzed e.g., D-glyceraldehyde 3-
phosphate: NAD oxidoreductase.
- IUBMB names unambiguous & informative, but
cumbersome in general use
Figure 5.1. Examples of the six major classes of the
international classification of enzymes (THF is tetrahydrofolate).
III. Properties of enzymes

• Enzymes are protein catalysts, increase velocity of a

chemical reaction, and not consumed
• Some types of RNA can act like enzymes, usually
catalyzing cleavage & synthesis of phosphodiester
bonds. RNAs with catalytic activity = ribozymes, less
common than protein catalysts
A. Active sites
- A special pocket, contains aa side chains that create a
3D surface complementary to S
- Active site binds S  ES complex that is converted to
EP  dissociates to E + P
Figure 5.2. Schematic representation of an enzyme with one active
site binding a substrate molecule.
B. Catalytic efficiency
- Most E catalyzed reactions are highly efficient,
103-108 x faster than uncatalyzed.
- Typically an E molecule  transforms ~ 100-1000
S molecules  P each second
- Number of S molecules  P per sec is “turnover
number”
C. Specificity
- E’s are highly specific, interacting with one or few
S & catalyze only one type of chemical reaction
D. Cofactors
- Some E’s associate with a non-protein cofactor
for activity
- e.g., metal ions (Zn2+ or Fe2+), and organic
molecules a.k.a co-enzymes, that are often
derivatives of vitamins e.g., NAD+ contains
niacin, FAD contains riboflavin, coenz A contain
pantothenic acid
- Holoenzymes = E + its cofactor
- Apoenzyme = protein portion of the holoenzyme
- In absence of appropriate cofactor, apoenz.
typically show no biologic activity
- Prosthetic group = tightly bound coenz that does
not dissociate from enz (e.g., biotin bound to
carboxylase)
E. Regulation
- E activity can be regulated i.e., E can be activated
or inhibited, i.e., rate of P formation responds to
needs of cell
F. Location within the cell
- Many E’s localized in specific organelles
- Compartmentalization isolates reaction S or P from
other competing reactions. This provides
favorable environ for reaction, & organizes the
1000’s of E’s in a cell into purposeful pathways
Figure 5.3. The intracellular
location of some important
biochemical pathways.
 How enzymes work

Mechanism of E action can be viewed from 2

different perspectives:
- Catalysis in terms of energy changes that occur
during reaction, i.e., E’s provide an alternate,
energetically favorable reaction pathway different
from uncatalyzed one
- How active site chemically facilitates catalysis
A. Energy changes occurring during the reaction
- All chemical reactions have an energy barrier
separating reactants and products = free energy
of activation  energy difference b/w reactants
and high energy intermediate that occurs during
formation of product
A ↔ T* ↔ B
- T* is the transition state = high energy
intermediate
Figure 5.4
Effect of an enzyme on
the activation energy of a
reaction
1. Free energy of activation:
- Difference in free energy b/w reactant and T*, because
of high free energy of activation, rates of uncatalyzed
chemical reactions are often slow
2. Rate of reaction:
- For molecules to react, they must contain sufficient
energy to overcome energy barrier of transition state
- In absence of E, only a small proportion of molecules
may possess enough energy to achieve T*. Rate of
reaction is determined by number of energized
molecules
- The lower free energy to pass through T*, & the faster
the reaction.
3. Alternate reaction pathway:
- An E allows a reaction to proceed under
conditions prevailing in cell by providing a
pathway with a lower free energy of activation
- E does not change free energies of R’s or P’s,
and so does not change equilibrium of reaction
B. Chemistry of active site
- Active site is a complex molecular machine
employing a diversity of chemical mechanisms
to facilitate R  P. A number of factors
responsible for catalytic efficiency of E’s e.g.,
1. Transition state stabilization: active site often
acts as a flexible molecular template that binds
S in a geometric structure resembling activated
T*. By stabilizing S in T*, the E greatly increases
the conc. of reactive intermediates that can be
converted to P, thus, accelerates reaction
2. Other mechanisms: active site can provide
catalytic groups that enhance probability that T*
is formed.
- In some E’s, groups can participate in general
acid-base catalysis e.g., aa residues provide or
accept protons
- In other E’s, catalysis may involve transient
formation of covalent enzyme-substrate complex
Enzymes that form covalent intermediates
 3. Visualization of the transition state

Figure 5.5. Schematic representation of energy changes accompanying formation

of enzyme-substrate complex and subsequent formation of a transition-state
complex.
V. Factors affecting reaction velocity

- E’s can be isolated and their properties studied

in vitro. Different E’s show different responses to
[S], Temp, pH
A. Substrate concentration
1. Maximal velocity: rate or velocity of a reaction
(v) is the # S molecules  P per unit time; it is
usually expressed as μmol of P formed per
minute. Rate of E-catalyzed reaction increases
with S conc. until a maximal velocity (Vmax) is
reached. Leveling off of reaction rate at high [S]
reflects saturation with S of all available binding
sites on E molecules present
2. Hyperbolic shape of the enzyme kinetics curve

- Most E’s show Michaelis-Menten kinetics, in

which plot of initial velocity, v0, against [S] is
hyperbolic
- In contrast, allosteric E’s frequently show
sigmoidal curve
Figure 5.6. Effect of
substrate conc. on
reaction velocity.
B. Temperature

1. Increase of velocity with temperature. As a

result of increased # of molecules having
sufficient energy to pass over energy barrier
and form P’s
2. Decrease of velocity with higher temperatures.
As a result of temp-induced denaturation of E.
Figure 5.7
Effect of temperature
on an enzyme-
catalyzed reaction.
C. pH

1. Effect of pH on ionization of active site: conc.

of H+ affects reaction velocity in several ways.
1st, catalytic process usually requires E and S
have specific chemical groups in ionized or
unionized state in order to interact e.g., amino
group of E be in protonated form (-NH3+). At
alkaline pH, this group is deprotonated and
rate of reaction declines
2. Effect of pH on E denaturation. Extremes of pH
can  denaturation, because structure of
catalytically active protein molecule depends
on ionic character of aa side chains
3. The pH optimum varies for different enzymes:
the pH at which maximal E activity is achieved is
different for different E’s, & often reflects [H+] at
which E functions in body e.g., pepsin, a
digestive E in stomach, is maximally active at pH
2, whereas other E’s, designed to work at
neutral pH are denatured by such an acidic
environ
Figure 5.8
Effect of pH on
enzyme-
catalyzed
reactions.
VI. Michaelis-Menten equation

A. Reaction model
- E reversibly combines with S to form ES complex
that subsequently breaks down to P,
regenerating free E. The model, involving one
S molecule: k1 k2
E + S ↔ ES → E + P
K-1

B. Michaelis-Menten equation
- Describes how reaction velocity varies with [S]
 V0 = initial velocity
Vmax [S] Vmax = maximal velocity
v0 = Km = Michaelis constant = (k-1 + k2)/k1
Km + [S] [S] = substrate conc

Assumptions made in deriving Michaelis-Menten eq:

1. Relative concentrations of E and S: [S] is much greater
than [E], so % of total S bound by E at any one time is
small
2. Steady-state assumption: [ES] does not change with time
i.e., rate of formation of ES = breakdown of ES (to E + S &
to E + P)
3. Initial velocity: only v0’s are used in analysis of E reactions.
i.e., rate of reaction is measured as soon as E and S are
mixed. At that time conc of P is very small and so, rate of
back reaction (P  S) can be ignored
C. Important conclusions about Michaelis-Menten
kinetics

1. Characteristics of Km:
- Km is characteristic of an E and its particular S, and
reflects affinity of E for that S.
- Km is numerically = [S] at which reaction velocity is ½
Vmax. Km does not vary with conc of E
a. Small Km: reflects a high affinity of E for S, as low
conc of S is needed to half-saturate the E- i.e., reach a
velocity that is ½ Vmax
b. Large Km: reflects a low affinity of E for S. As high [S]
is needed to half saturate the E.
Figure 5.9
Effect of substrate
concentration on reaction
velocities for two enzymes:
enzyme 1 with a small Km,
and enzyme 2 with a large
Km.
2. Relationship of velocity to enzyme concentration

- Rate of reaction α [E] at all S conc’s. e.g., if [E] is

halved, initial rate of reaction (v0), & that of
Vmax, are reduced to ½ that of the original.
- Order of reaction:
- When [S] is much less than Km, velocity of reaction is
~ proportional to [S]. Rate of reaction is said to be 1st
order wrt S.
- When [S] is much greater than Km, velocity is
constant and = Vmax. Rate of reaction is then
independent of [S], and is said to be zero order wrt
[S].
Figure 5.10
Effect of substrate concentration on
reaction velocity for an enzyme
catalyzed reaction.
D. Lineweaver-Burke plot

- When v0 is plotted against S, it is not always

possible to determine when Vmax has been
achieved because of the gradual upward slope
of the hyperbolic curve at high [S]
- If 1/v0 is plotted vs. 1/[S], a straight line is
obtained. This plot = Lineweaver-Burke plot
(a.k.a double-reciprocal plot) can be used to
calculate Km & Vmax, as well as to determine
mechanism of action of E inhibitors
1. The eq. describing Lineweaver-Burke plot is:

Km 1
1/v0 = +
Vmax [S] Vmax

- Where intercept on x-axis = -1/Km, & intercept

on y-axis = 1/Vmax
Figure 5.11. Lineweaver-Burke plot.
VII. Inhibition of enzyme activity

• Any S that can diminish velocity of E catalyzed

reaction is inhibitor (I)
• Reversible inhibitors bind to E through non-
covalent bonds. Dilution of E-I complex results in
dissociation of reversibly bound I, and recovery
of E activity
• Irreversible inhibition occurs when an inhibited E
does not regain activity on dilution of E-I
complex
• Commonly encountered types:
– Competitive
– Non-competitive
 A. Competitive inhibition
• I binds reversibly to same site of S, i.e., competes with S
for that site
1. Effect on Vmax: effect of competitive I is reversed by
increasing [S]. At sufficiently high [S], reaction velocity
reaches Vmax observed in absence of I.
2. Effect on Km: competitive I increases apparent Km for a
given S. i.e., in presence of competitive I, more S is
needed to achieve ½ Vmax
3. Effect on Lineweaver-Burke plot: plots of inhbited &
uninhibited reactions intersect on y-axis at 1/Vmax
(Vmax is unchanged). Inhibited and uninhibited reactions
show different x-axis intercepts i.e., apparent Km is
increased in presence of competitive I.
Figure 5.12. A. Effect of a competitive inhibitor on the reaction velocity (vo) versus
substrate [S] plot. B. Lineweaver-Burke plot of competitive inhibition of an enzyme.
4. Statin drugs as examples of competitive inhibitors:
- This group of antihyperlipidemic agents
competitively inhibits 1st committed step in
cholesterol synthesis
- This reaction is catalyzed by hydroxymethylglutaryl
CoA reductase (HMG CoA reductase)
- Statin drugs e.g., atorvastatin (Lipitor) & simvastatin
(Zocor) are structural analogs of natural S for this
E, & compete effectively to inhibit HMG CoA
reductase  inhibit de novo cholesterol synthesis,
thereby lowering plasma cholesterol levels.
Figure 5.13. Lovastatin
competes with HMG CoA for
the active site of HMG
CoA reductase.
B. Non-competitive inhibition

- Occurs when I and S bind at different sites on

E. The non-competitive I can bind either free E
or ES complex, thereby preventing reaction
from occurring
1. Effect on Vmax: non-competitive inhibition can
not be overcome by increasing conc of S, i.e.,
non-competitive inhibition decreases Vmax
2. Effect on Km: non-competitive I’s do not
interfere with binding of S to E. So, E shows
same Km in presence or absence of the non-
competitive inhibitor
3. Effect on Lineweaver-Burke plot: non-competitive
inhibition is differentiated by noting Vmax decrease,
whereas Km is unchanged in presence of non-
competitive inhibitor
4. Examples of non-competitive inhibitors: some I’s act by
forming covalent bonds with specific groups of E’s. e.g.,
lead forms covalent bonds with sulfhydryl side chains of
cysteine in proteins. Ferrochelatase catalyzes insertion
of Fe2+ into protoporphyrin (a precursor of heme) is
sensitive to inhibition by lead.
Other e.g.’s are certain insecticides whose neurotoxic
effects result from their irreversible binding at catalytic
site of acetylcholiesterase (that cleaves the
neurotransmitter acetylcholine)
Figure 5.14. A. Effect of a noncompetitive inhibitor on the reaction velocity (vo)
versus substrate [S] plot. B. Lineweaver-Burke plot of noncompetitive inhibition of an
enzyme.
C. Enzyme inhibitors as drugs

• E.g., the widely prescribed ß-lactam antibiotics

e.g., penicillin & amoxycillin inhibit enzymes
involved in bacterial CW synthesis
• Drugs may also act by inhibiting extracellular
reactions e.g., angiotensin-converting enzyme
(ACE) inhibitors. They lower blood pressure by
blocking the E that cleaves angiotensin I to form
the potent vasoconstrictor, angiotensin II. These
drugs, e.g., captopril, enalapril, lisinopril, cause
vasodilation & so reduction in blood pressure
Figure 5.15
A noncompetitive
inhibitor binding to
both free enzyme and
enzyme-substrate
complex.
VIII. Regulation of enzyme activity

• Regulation of reaction velocity of E’s is essential

to coordinate numerous metabolic processes
• Rate of most E’s responsive to changes in [S],
as intracellular level of many S’s is in range of
Km. an increase in [S]  increase in reaction
rate  return [S] to normal.
• Some E’s with specialized regulatory functions
respond to allosteric effectors or covalent
modification, or show altered rates of E
synthesis when physiologic conditions are
changed
A. Allosteric binding sites

• Allosteric E’s regulated by molecules = effectors (also

modifiers), that bind non-covalently at a site other than
active site
• These E’s are composed of multiple subunits, &
regulatory site may be present on a subunit that is not
itself catalytic
• Presence of allosteric effector can alter affinity of E to its
S, or modify maximal catalytic activity of E, or both
• Effectors that inhibit E activity = negative effectors, that
increase E activity = positive effectors
• Allosteric E’s usually contain subunits and frequently
catalyze the committed step early in a pathway
1. Homotropic effectors: when S itself serves as
effector, effect is said to be homotropic. Most
often allosteric S functions as a positive effector
- Presence of S molecule at one site enhances
catalytic properties of other S-binding sites i.e.,
binding sites exhibit cooperativity
- These E’s show sigmoidal curve when reaction
velocity (v0) is plotted agains [S]
- Positive & negative effectors of allosteric E’s can
affect either Vmax or Km, or both
Figure 5.16. Effects of negative or positive effectors on an allosteric
enyzme. A. Vmax is altered. B. The substrate concentration that gives half
maximal velocity (K0.5) is altered.
2. Heterotropic effectors: effector different from S, effect =
heterotropic.
-e.g., consider the following feedback inhibition

- E that converts A to B has an allosteric site that binds the

end-product. If conc. of end product increases (e.g., not
used as rapidly as synthesized), the initial enz in the
pathway is inhibited
- Feedback inhibition provides cell product it
needs by regulating flow of S molecules through
the pathway that synthesizes the product
- e.g., glycolytic E phosphofructokinase is
allosterically inhibited by citrate, which is not a S
for the E.
B. Regulation of enzymes by covalent modification

• Many E’s may be regulated by covalent

modification, frequently addition or removal of
phosphate groups from specific ser, thr, or tyr
residues
• Protein phosphorylation is recognized as one of
primary ways in which cell processes regulated
1. Phosphorylation & dephosphorylation: phospho.
reactions are catalyzed by a family of E’s =
protein kinases that use ATP as P donor. P
groups are cleaved by phosphoprotein
phosphatases
Figure 5.18. Covalent modification by the addition and
removal of phosphate groups.
2. Response of enzyme to phosphorylation:
depending on specific E, the P-form may be
more or less active than unphospho-form. e.g.,
P-form of glycogen phosphorylase (degrades
glycogen) has increased activity, whereas
addition of P to glycogen synthase (synthesizes
glycogen) decreases activity
C. Induction & repression of enzyme synthesis

• Cells can also regulate amount of E present- usually by

altering rate of E synthesis.
• Increased (induction) or decreased (repression) of E
synthesis leads to an alteration in the total population of
active sites
• Efficiency of existing E molecules is not affected
• E’s subject to regulation of synthesis are often those
needed at one stage of development or under selected
physiologic conditions
• E.g., elevated levels of insulin as a result of high blood
glucose levels can cause an increase in synthesis of key
enzymes involved in glucose metabolism
- In contrast, E’s that are in constant use are
usually not regulated by altering rate of
synthesis
- Alterations in E levels by induction or repression
of protein synthesis are slow (hours to days),
compared with allosterically regulated changes
in E activity, which occur in seconds to minutes.
Figure 5.19. Mechanisms for regulating enzyme activity.
IX. Enzymes in clinical diagnosis
• Plasma E’s can be classified into 2 major groups
- 1st a relatively small group of E’s are actively secreted
into blood by certain cell types e.g., liver secretes
zymogens (inactive precursors) of E’s involved in
coagulation
- 2nd a large # of E species are released from cells during
normal cell turnover. These E’s almost always function
intracellularly, & no physiologic use in plasma
• In healthy individuals, levels of these E’s are fairly
constant, and represent a steady in which rate release
from damaged cells into plasma is balanced by equal
rate of removal of the E protein from plasma
- Presence of elevated E activity in plasma may
indicate tissue damage that is accompanied by
increased release of intracellular E’s.
Note:
- Plasma is fluid, non-cellular part of blood.
- Lab assays of E activity most often use serum,
obtained by centrifugation of whole blood after it
has been allowed to coagulate.
- Plasma is a physiologic fluid, serum is prepared
in lab
Figure 5.20. Release of enzymes from normal and diseased or traumatized cells
A. Alteration of plasma enzyme levels in disease
states
- Many diseases that cause tissue damage 
increased release of intracellular E’s into
plasma
- Activities of many of these E’s routinely
determined for diagnostic purposes in
diseases of heart, liver, skeletal muscle, etc.
- Level of specific E activity in plasma frequently
correlates with extent of tissue damage
- So, determining degree of elevation of an E
activity in plasma is often useful in evaluating
prognosis for patient
B. Plasma enzymes as diagnostic tools
- Some E’s show relatively high activity in only
one or few tissues. Presence of such E’s in
plasma : damage to corresponding tissue.
- E.g., alanine aminotransferase (ALT) is
abundant in liver. Appearance of elevated levels
of ALT in plasma signals possible damage to
hepatic tissue
- Increases in plasma levels of E’s with a wide
tissue distribution provide a less specific
indication of site of cellular injury. Lack of tissue
specificity limits diagnostic value of many E’s
C. Isoenzymes and diseases of the heart
- Most isozymes are E’s that catalyze same reaction. But,
they do not necessarily have same physical properties
because of genetically determined differences in aa
sequence
- Isoenzymes different numbers of charged aa’s, and may
be separated from each other by electrophoresis
- Different organs frequently contain characteristic
proportions of different isozymes
- Pattern of isozymes found in plasma may serve as
means to identify site of tissue damage. E.g., plasma
levels of creatine kinase (CK) & lactate dehydrogenase
(LDH) commonly determined in diagnosis of myocardial
infarction. Particularly useful when electrocardiogram is
difficult to interpret, e.g., when there have been previous
episodes of heart disease
1. Quaternary structure of isoenzymes
- Many isozymes contain different subunits in
various combinations. e.g., CK occurs as 3
isozymes. Each is a dimer composed of 2
polyp’s (B & M) subunits associated in 1 of 3
combinations: CK1 = BB, CK2 = MB, CK3 =
MM. each CK shows a characteristic
electrophoretic mobility
Figure 5.21
Subunit structure and electrophoretic
mobility and enzyme activity of
creatine kinase isoenzymes.
2. Diagnosis of myocardial infarction:
- Myocardial muscle is the only tissue that contains > 5%
of total CK activity as CK2 (MB) isozyme.
- Appearance of this hybrid isozyme in plasma is virtually
specific for infarction of the myocardium
- Following an acute MI, this isozyme appears ~ 4-8 hours
following onset of chest pain & reaches a peak of activity
at ~ 24 h
NOTE: Lactate dehydrogenase (LDH) activity is also
elevated in plasma following infarction, peaking 36-40 h
after onset of symptoms
- LDH activity is, thus, of diagnostic value in patients
admitted > 48 h after infarction- a time when plasma CK2
may provide equivocal results
Figure 5.22
Appearance of
creatine kinase (CK)
and lactate
dehydrogenase (LDH)
in plasma after
a myocardial
infarction.
3. Newer markers for MI:
- Troponin T and Troponin I are regulatory
proteins involved in myocardial contractility
- They are released into plasma in response to
cardiac damage
- Elevated serum troponins are more predictive of
adverse outcomes in unstable angina or MI than
conventional assay of CK2
 Summary
• E’s = protein catalysts, increase v of chemical reactions
by lowering energy of transition state, not consumed
• E’s contain special pocket = active site that has aa side
chains which create 3D surface complementary to S.
Active site binds S  ES complex  EP  E + P
• E’s allow reactions to proceed rapidly under conditions
prevailing in cells by providing alternate reaction path
with lower free energy of activation
• E’s do not change free energy of reactants or products,
so do not change equilibrium of reaction
• Most E’s show Michaelis-Menten kinetics, a plot of v0 vs.
[S]  hyperbolic shape
• Any substance that can diminish v of E-catalyzed
reaction is inhibitor.
• Two common types: competitive (increases apparent
Km) & non-competitive (decreases Vmax)
• Multi-subunit allosteric E’s frequently show a
sigmoidal curve
– Frequently catalyze committed (rate limiting) step(s)
of a pathway
– Allosteric E’s are regulated by effectors (modifiers)
that bind non-covalently at a site other than active
site. Effectors can be +ve (accelerate E-catalyzed
reaction) or –ve (slow down)
– An allosteric effector can alter affinity of E for its S, or
modify maximal catalytic activity of E, or both.
Figure 5.23
Key concept map
for the enzymes. S
= substrate, [S] =
substrate
concentration,
P = product, E =
enzyme, vo = initial
velocity, Vmax =
maximal velocity,
Km = Michaelis
constant.