CHEM 4311

General Biochemistry I
Fall 2012

Chapter 13-15
Enzyme Chapter 13
EXAM - October 19 Enzyme Kinetics

Instructor: Dr. Khairul I Ansari

Office: 316CPB
Phone: 817-272-0616
email: kansari@uta.edu
Office hours 12 am – 1:30 pm Tuesday &.Thursday

Virtually All Reactions in Cells Are Mediated by Virtually All Reactions in Cells Are Mediated by
Enzymes Enzymes

• Enzymes catalyze thermodynamically favorable

reactions, causing them to proceed at extraordinarily
rapid rates (see Figure 13.1)
• Enzymes provide cells with the ability to exert kinetic
control over thermodynamic potentiality
• Living systems use enzymes to accelerate and control
the rates of vitally important biochemical reactions
• Enzymes are the agents of metabolic function

Figure 13.1 Reaction profile showing the large free energy of activation for
glucose oxidation. Enzymes lower ∆G‡, thereby accelerating rate.

13.1 What Characteristic Features Define 13.1 What Characteristic Features Define
Enzymes? Enzymes?

• Catalytic power is defined as the ratio of the enzyme- • Enzymes can accelerate reactions as much as 1021
catalyzed rate of a reaction to the uncatalyzed rate over uncatalyzed rates
• Specificity is the term used to define the selectivity of
enzymes for their substrates • Urease is a good example:
• Regulation of enzyme activity ensures that the rate of – Catalyzed rate: 3x104/sec
metabolic reactions is appropriate to cellular requirements
– Uncatalyzed rate: 3x10 -10/sec
• Enzyme nomenclature provides a systematic way of naming
metabolic reactions – Ratio (catalytic power) is 1x1014
• Coenzymes and cofactors are nonprotein components
essential to enzyme activity.
 Specificity Enzymes are the Agents of Metabolic Function

• Enzymes selectively recognize proper substrates

over other molecules Figure 13.2 The breakdown of
• Enzymes produce products in very high yields - glucose by glycolysis provides a
often much greater than 95% prime example of a metabolic
pathway.
• Specificity is controlled by structure - the unique
fit of substrate with enzyme controls the
selectivity for substrate and the product that’s
formed.

Enzyme Nomenclature Provides a Systematic Way of

90% yield in each step; 35% over 10 steps Naming Metabolic Reactions

Figure 13.3 Yields in biological reactions must be substantially greater

than 90%.

Coenzymes and Cofactors Are Nonprotein
Components Essential to Enzyme Activity
13.2 Can the Rate of an Enzyme-Catalyzed Reaction Be
Defined in a Mathematical Way?
• Kinetics is the branch of science concerned with the rates of
reactions
• Enzyme kinetics seeks to determine the maximum reaction
velocity that enzymes can attain and the binding affinities for
substrates and inhibitors
• Analysis of enzyme rates yields insights into enzyme
mechanisms and metabolic pathways
• This information can be exploited to control and manipulate
the course of metabolic events
 Chemical Kinetics Provides a Foundation for Exploring
Several Kinetics Terms to Understand Enzyme Kinetics

• rate or velocity • Consider a reaction of overall stoichiometry as

shown:
• rate constant A→ P
• rate law
• order of a reaction d[P] −d[ A]
v= =
• molecularity of a reaction dt dt
−[ A]
v= = k[ A]
dt
• The rate is proportional to the concentration
of A

Chemical Kinetics Provides a Foundation for

Exploring Enzyme Kinetics
The Time-Course of a First-Order Reaction
• The simple elementary reac@on of A→P is a first-
order reaction
• Figure 13.4 shows the course of a first-order reaction
as a function of time
• This is a unimolecular reaction
• For a bimolecular reaction, the rate law is:
v = k[A][B]
• Kinetics cannot prove a reaction mechanism
• Kinetics can only rule out various alternative
hypotheses because they don’t fit the data Figure 13.4 Plot of the course of a first-order reaction. The
half-time, t1/2 is the time for one-half of the starting amount of
A to disappear.

Catalysts Lower the Free Energy of Activation for a Catalysts Lower the Free Energy of Activation for a
Reaction Reaction
• A typical enzyme-catalyzed reaction must pass through a
transition state
• The transition state sits at the apex of the energy profile
in the energy diagram
• The reaction rate is proportional to the concentration of
reactant molecules with the transition-state energy
• This energy barrier is known as the free energy of
activation
• Decreasing ∆G‡ increases the reaction rate
• The activation energy is related to the rate constant by: Figure 13.5 Energy diagram for a chemical reaction (A→P)
and the effects of (a) raising the temperature from T1 to T2, or
(b) adding a catalyst.
k = Ae− ∆G/ RT
 13.3 What Equations Define the Kinetics of
The Transition State
Enzyme-Catalyzed Reactions?

Understand the difference between ∆G and ∆G‡
• The overall free energy change for a reaction, ∆G, is
related to the equilibrium constant
• The free energy of activation for a reaction, ∆G‡, is
related to the rate constant
• It is extremely important to appreciate this distinction
• Simple first-order reactions display a plot of the reaction
rate as a function of reactant concentration that is a
straight line (Figure 13.6)
• Enzyme-catalyzed reactions are more complicated
• At low concentrations of the enzyme substrate, the rate is
proportional to S, as in a first-order reaction
• At higher concentrations of substrate, the enzyme
reaction approaches zero-order kinetics
• This behavior is a saturation effect

13.3 What Equations Define the Kinetics of As [S] increases, kinetic behavior changes from 1st
Enzyme-Catalyzed Reactions? order to zero-order kinetics

Figure 13.7 Substrate saturation

curve for an enzyme-catalyzed
Figure 13.6 A plot of υ versus [A] for the unimolecular chemical reaction, reaction.
A→P, yields a straight line having a slope equal to k. This reaction is a first-
order reaction.

The Michaelis-Menten Equation is the Fundamental [ES] Remains Constant Through Much of the Enzyme Reaction
Equation of Enzyme Kinetics Time Course in Michaelis-Menten Kinetics

Louis Michaelis and Maud Menten's theory Figure 13.8 Time course for a
typical enzyme-catalyzed reaction
• assumes the formation of an enzyme-substrate
complex obeying the Michaelis-Menten,
Briggs-Haldane models for
• assumes that the ES complex is in rapid equilibrium enzyme kinetics. The early state
with free enzyme
of the time course is shown in
• assumes that the breakdown of ES to form products greater magnification in the
is slower than bottom graph.
1) formation of ES and
2) breakdown of ES to re-form E and S
 The Michaelis-Menten equation Understanding Km
The "kinetic activator constant"
V [S] • Km is a constant
v = max • Km is a constant derived from rate constants
K m + [S] • Km is, under true Michaelis-Menten conditions, an
estimate of the dissociation constant of E from S
• Small Km means tight binding; high Km means weak
where Vmax = k2 [E T ] binding

and Km =
( k−1 + k2 )
k1

Understanding Vmax The dual nature of the Michaelis-Menten equation

The theoretical maximal velocity Combination of 0-order and 1st-order kinetics

• Vmax is a constant
• Vmax is the theoretical maximal rate of the reaction -
but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is asymptotically approached as substrate is
increased
• When S is low, the equation for rate is 1st order in S
• When S is high, the equation for rate is 0-order in S
• The Michaelis-Menten equation describes a rectangular
hyperbolic dependence of v on S
• See Figure 13.7
• The relation of the “rectangular hyperbola” to the enzyme
kinetics profile is described in references at the end of the
chapter

The Turnover Number Defines the Activity of One

Table 13.3 gives the Km values for some enzymes and Enzyme Molecule
their substrates

A measure of catalytic activity

• kcat, the turnover number, is the number of substrate
molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec to many
millions per sec
 The Turnover Number Defines the Activity of One The Ratio kcat/Km Defines the Catalytic Efficiency of an
Enzyme Molecule Enzyme

The catalytic efficiency: kcat/Km

An estimate of "how perfect" the enzyme is
• kcat/Km is an apparent second-order rate constant
• It measures how well the enzyme performs when S is
low
• The upper limit for kcat/Km is the diffusion limit - the
rate at which E and S diffuse together

The Ratio kcat/Km Defines the Catalytic Efficiency of an Linear Plots Can Be Derived from the Michaelis-
Enzyme Menten Equation
Be able to derive these equations
• Lineweaver-Burk:
• Begin with v = Vmax[S]/(Km + [S]) and take the reciprocal of
both sides
• Rearrange to obtain the Lineweaver-Burk equation:

1  Km   1  1
=    +
v  Vmax   [S]  Vmax
• A plot of 1/v versus 1/[S] should yield a straight line

Linear Plots Can Be Derived from the Michaelis- Linear Plots Can Be Derived from the Michaelis-
Menten Equation Menten Equation

• Hanes-Woolf:
• Begin with Lineweaver-Burk and multiply both sides
by [S] to obtain:

[S]  1  Km
= [S] +
v  Vmax  Vmax
• Hanes-Woolf is best - why?
• Because Hanes-Woolf has smaller and more
Figure 13.9 The Lineweaver- consistent errors across the plot
Burk double-reciprocal plot.
 Linear Plots Can Be Derived from the Michaelis-Menten
Equation
Figure 13.11 A Hanes-Woolf
plot of [S]/v versus [S]
Enzymatic Activity is Strongly Influenced by pH
• Enzyme-substrate recognition and catalysis are
greatly dependent on pH
• Enzymes have a variety of ionizable side chains that
determine its secondary and tertiary structure and
also affect events in the active site
• The substrate may also have ionizable groups
• Enzymes are usually active only over a limited range
of pH
• The effects of pH may be due to effects on Km or Vmax
or both

The Response of Enzymatic Activity to Temperature is

Enzymatic Activity is Strongly Influenced by pH
Complex

• Rates of enzyme-catalyzed reactions generally

increase with increasing temperature
• However, at temperatures above 50°to 60°C,
enzymes typically show a decline in activity
• Two effects here:
– Enzyme rate typically doubles in rate for ever 10º
C as long as the enzyme is stable and active
– At higher temperatures, the protein becomes
Figure 13.11 The pH activity profiles of four different enzymes. unstable and denaturation occurs

The Response of Enzymatic Activity to Temperature is 13.4 What Can Be Learned from the Inhibition of
Complex Enzyme Activity?

• Enzymes may be inhibited reversibly or

irreversibly
• Reversible inhibitors may bind at the active site or
at some other site
• Enzymes may also be inhibited in an irreversible
manner
• Penicillin is an irreversible suicide inhibitor

Figure 13.12 The effect

of temperature on
enzyme activity.
Reversible Inhibitors May Bind at the Active Site or at Competitive Inhibitors Compete With Substrate for the
Some Other Site Same Site on the Enzyme

Figure 13.13 Lineweaver-Burk plot of competitive inhibition,

showing lines for no I, [I], and 2[I].

Succinate Dehydrogenase – a Classic Example of Pure Noncompetitive Inhibition – where S and I bind
Competitive Inhibition to different sites on the enzyme

Figure 13.15 Lineweaver-Burk plot of pure noncompetitive

inhibition. Note that I does not alter Km but that it decreases
Vmax.
Figure 13.14 Structures of succinate, the substrate of succinate
dehydrogenase (SDH), and malonate, the competitive inhibitor.
Fumarate (the product of SDH) is also shown.

Mixed Noncompetitive Inhibition: binding of I by E Uncompetitive Inhibition, where I combines only with
influences binding of S by E E, but not with ES

Figure 13.17 Lineweaver-Burk plot of

uncompetitive inhibition. Note that
both intercepts change but the slope
(Km/Vmax) remains constant in the
presence of I.

Figure 13.16 Lineweaver-Burk plot of mixed noncompetitive

inhibition. Note that both intercepts and the slope change in
the presence of I.
 13.5 - What Is the Kinetic Behavior of Enzymes
Enzymes Can Be Inhibited Irreversibly Catalyzing Bimolecular Reactions?
• Enzymes often catalyze reactions involving two (or
more) substrates
Figure 13.18 Penicillin is
an irreversible inhibitor of • Bisubstrate reactions may be sequential or single-
the enzyme glycoprotein displacement reactions or double-displacement (ping-
peptidease, which pong) reactions
catalyzes an essential step • Single-displacement reactions can be of two distinct
in bacterial cell all
classes:
synthesis.
1. Random, where either substrate may bind
first, followed by the other substrate
2. Ordered, where a leading substrate binds
first, followed by the other substrate

13.5 - What Is the Kinetic Behavior of Enzymes Viagra – An Unexpected Outcome in a Program of
Catalyzing Bimolecular Reactions? Drug Design

Note the similarities in the structures of cGMP (left) and

Viagra (right). Viagra produces an increase in [cGMP] in
penile vascular tissue, allowing vascular muscle relaxation,
improved blood flow, and erection.
Figure 13.19 Single-deplacement bisubstrate mechanism.

Conversion of AEB to PEQ is the Rate-Limiting Step in Creatine Kinase Acts by a Random, Single-
Random, Single-Displacement Reactions Displacement Mechanism

In this type of sequential reaction, all possible binary The overall direction of the reaction will be determined by
enzyme-substrate and enzyme-product complexes are the relative concentrations of ATP, ADP, Cr, and CrP and the
formed rapidly and reversibly when enzyme is added to a equilibrium constant for the reaction.
reaction mixture containing A, B, P, and Q.
 Creatine Kinase Acts by a Random, Single- In an Ordered, Single-Displacement Reaction, the
Displacement Mechanism Leading Substrate Must Bind First

Figure 13.21 The structures

of creatine and creatine
phosphate, guanidinium
compounds that are
important in muscle energy
metabolism.
The leading substrate (A) binds first, followed by B.
Reaction between A and B occurs in the ternary complex
and is usually followed by an ordered release of the
products, P and Q.

An Alternative way of Portraying the Ordered, Single- The Double Displacement (Ping-Pong)
Displacement Reaction Reaction

Double-Displacement (Ping-Pong) reactions proceed via

formation of a covalently modified enzyme intermediate.
Reactions conforming to this kinetic pattern are characterized
by the fact that the product of the enzyme’s reaction with A
(called P in the above scheme) is released prior to reaction of
the enzyme with the second substrate, B.

An Alternative Presentation of the Double- The Double Displacement (Ping-Pong)

Displacement (Ping-Pong) Reaction Reaction

Figure 13.22
Double-
displacement
(ping-pong)
bisubstrate
mechanisms are
characterized by
parallel lines.
 Glutamate:aspartate Aminotransferase 13.7 – How Can Enzymes Be So Specific?
• The “Lock and key” hypothesis was the first explanation
for specificity
• The “Induced fit” hypothesis provides a more accurate
description of specificity
• Induced fit favors formation of the transition state
Figure 13.23
An enzyme • Specificity and reactivity are often linked. In the
conforming to a hexokinase reaction, binding of glucose in the active site
double-
displacement induces a conformational change in the enzyme that
bisubstrate causes the two domains of hexokinase to close around
mechanism.
the substrate, creating the catalytic site

13.7 – How Can Enzymes Be So Specific? 13.7 – Are All Enzymes Proteins?

• Ribozymes - segments of RNA that display enzyme

activity in the absence of protein
– Examples: RNase P and peptidyl transferase
• Abzymes - antibodies raised to bind the transition
state of a reaction of interest
– For a good review of abzymes, see Science, Vol.
269, pages 1835-1842 (1995)
– Transition states are covered in more depth in in
Figure 13.24 A drawing, roughly to scale, of H2O, glycerol, Chapter 14
glucose, and an idealized hexokinase molecule. Binding of
glucose in the active site induces a conformational change
in the enzyme that causes the two domains of hexokinase
to close around the substrate, creating the catalytic site.

RNA Molecules That Are Catalytic Have Been Termed RNA Molecules That Are Catalytic Have Been
“Ribozymes” Termed “Ribozymes”

Figure 13.26 (a) The 50S subunit from H. marismortui. (b) The
aminoacyl-tRNA (yellow) and the peptidyl-tRNA (orange) in the
peptidyl transferase active site.
Figure 13.25 RNA splicing in
Tetrahymena rRNA maturation.
RNA Molecules That Are Catalytic Have Been Termed
Antibody Molecules Can Have Catalytic Activity
“Ribozymes”

Figure 13.28 (a) Intramolecular hydrolysis of a hydroxy ester

yields a δ-lactone.

(b) The cyclic

phosphonate ester
analog of the cyclic
transition state.
Figure 13.27 The peptidyl transferase reaction.

13.8 Is It Possible to Design An Enzyme to Catalyze 13.8 Is It Possible to Design An Enzyme to Catalyze Any
Any Desired Reaction? Desired Reaction?

• A known enzyme can be “engineered” by in vitro

mutagenesis, replacing active site residues with new
ones that might catalyze a desired reaction
• Another approach attempts to design a totally new
protein with the desired structure and activity
– This latter approach often begins with studies “in Figure 13.29 cis-1,2-Dichloroethylene (DCE) is an
silico” – i.e., computer modeling industrial solvent that poses hazards to human health.
– Protein folding and stability issues make this
Site-directed mutations have enabled the conversion of a
approach more difficult
bacterial epoxide hydrolase to catalyze the chlorinated
– Further, the cellular environment may provide epoxide hydrolase reaction.
complications not apparent in the computer
modeling

14.1 What Are the Magnitudes of Enzyme-Induced

Rate Accelerations?

• Enzymes are powerful catalysts

• The large rate accelerations of enzymes (107 to 1015)
Chapter 14 correspond to large changes in the free energy of
Mechanisms of Enzyme Action activation for the reaction
• All reactions pass through a transition state on the
reaction pathway
• The active sites of enzymes bind the transition state
of the reaction more tightly than the substrate
• By doing so, enzymes stabilize the transition state
and lower the activation energy of the reaction
14.1 What Are the Magnitudes of Enzyme-Induced Rate 14.2 What Role Does Transition-State Stabilization Play
Accelerations? in Enzyme Catalysis?
• The catalytic role of an enzyme is to reduce the energy
barrier between substrate S and transition state X‡
• Rate acceleration by an enzyme means that the energy
barrier between ES and EX‡ must be smaller than the
barrier between S and X‡
• This means that the enzyme must stabilize the EX‡
transition state more than it stabilizes ES
• See Eq. 14.3

14.2 What Role Does Transition-State Stabilization Play 14.2 What Role Does Transition-State Stabilization
in Enzyme Catalysis? Play in Enzyme Catalysis?

Competing effects determine the position of ES on the

energy scale
• Try to mentally decompose the binding effects at the
active site into favorable and unfavorable
• The binding of S to E must be favorable
• But not too favorable!
• Km cannot be "too tight" - goal is to make the energy
barrier between ES and EX‡ small

Figure 14.1 Enzymes catalyze reactions by lowering the activation

energy. Here the free energy of activation for (a) the uncatalyzed
reaction is larger than that of the enzyme-catalyzed reaction.

14.3 How Does Destabilization of ES Affect Enzyme 14.3 How Does Destabilization of ES Affect Enzyme
Catalysis? Catalysis?
Raising the energy of ES raises the rate

• For a given energy of EX‡, raising the energy of ES

will increase the catalyzed rate
• This is accomplished by
a) loss of entropy due to formation of ES
b) destabilization of ES by
• strain
• distortion
• desolvation
Figure 14.2 The intrinsic binding energy of ES is compensated by entropy
loss due to binding of E and S and by destabilization due to strain and
distortion.
 14.3 How Does Destabilization of ES Affect Enzyme 14.3 How Does Destabilization of ES Affect
Catalysis? Enzyme Catalysis?

Figure 14.4 (a) Formation of the ES complex results in entropy

loss. The ES complex is a more highly ordered, low-entropy
Figure 14.3 (a) Catalysis does not occur if ES and X‡ are equally state for the substrate.
stabilized. (b) Catalysis will occur if X‡ is stabilized more than ES.

14.3 How Does Destabilization of ES Affect Enzyme 14.3 How Does Destabilization of ES Affect
Catalysis? Enzyme Catalysis?

Figure 14.4 (b) Substrates typically lose waters of hydration Figure 14.4 (c) Electrostatic destabilization of a substrate may
in the formation in the formation of the ES complex. arise from juxtaposition of like charges in the active site. If
Desolvation raises the energy of the ES complex, making it charge repulsion is relieved in the reaction, electrostatic
more reactive. destabilization can result in a rate increase.

14.4 How Tightly Do Transition-State Analogs Bind to 14.4 How Tightly Do Transition-State Analogs Bind to
the Active Site? the Active Site?

Very tight binding to the active site

• The affinity of the enzyme for the transition state may
be 10 -20 to 10-26 M!
• Can we see anything like that with stable molecules?
• Transition state analogs (TSAs) are stable molecules
that are chemically and structurally similar to the
transition state
• Proline racemase was the first case
• See Figure 14.6 for some recent cases
Figure 14.5 The proline racemase reaction. Pyrrole-2-
carboxylate and ∆-1-pyrroline-2-carboxylate mimic the planar
transition state of the reaction.
14.4 How Tightly Do Transition-State Analogs Bind to 14.4 How Tightly Do Transition-State Analogs Bind to
the Active Site? the Active Site?

(b) Purine riboside inhibits adenosine deaminase. The

Figure 14.6 (a) Phosphoglycolohydroxamate is an analog of the hydrated form is an analog of the transition state of the
enediolate transition state of the yeast aldolase reaction. reaction.

Transition-State Analogs Make Our World

Transition-State Analogs Make Our World Better
Better
Blood pressure is partly regulated
• Enzymes are often targets for drugs and other by aldosterone, a steroid made and
beneficial agents released in blood vessels by
• Transition-state analogs often make ideal enzyme angiotensin II, a peptide produced
inhibitors from angiotensinogen in two
proteolytic steps by renin and ACE.
• Enalapril and Aliskiren lower blood pressure Enalapril is an ACE inhibitor.
• Statins lower serum cholesterol Aliskiren is a renin inhibitor. Both
are TSAs.
• Protease inhibitors are AIDS drugs
• Juvenile hormone esterase is a pesticide target
• Tamiflu is a viral neuraminidase inhibitor

Transition-State Analogs Make Our World Better Transition-State Analogs Make Our World Better

Statins such as Lipitor are

powerful cholesterol-lowering
drugs, because they are
transition-state analog inhibitors
of HMG-CoA reductase, a key
Statins such as Lipitor are
enzyme in the biosynthetic
powerful cholesterol-lowering
pathway for cholesterol.
drugs, because they are
transition-state analog inhibitors
of HMG-CoA reductase, a key
enzyme in the biosynthetic
pathway for cholesterol.
Transition-State Analogs Make Our World Better Transition-State Analogs Make Our World Better

Insects have significant effects on human health. Malaria,

West Nile virus, and viral encephalitis are carried by
Invirase (saquinavir) by Roche and similar “protease inhibitor” mosquitoes (left). Lyme disease and Rocky Mountain spotted
drugs are transition-state analogs for the HIV-1 protease. fever are carried by ticks (right).

The 1918 flu pandemic killed more than 20 million

Transition-State Analogs Make Our World Better people worldwide.

One strategy for controlling insect populations is to alter the

actions of juvenile hormone, a terpene-based substance that
regulates insect life cycle processes. Levels of juvenile
hormone are controlled by juvenile hormone esterase (JHE),
and inhibition of JHE is toxic to insects. OTEP (figure) is a
potent transition state-analog inhibitor of JHE.

How many other drug targets might there be?

Tamiflu is a Viral Neuraminidase Inhibitor
• Influenza is a serious illness that
affects 5% to 15% of the earth’s • The human genome contains approximately
population each year and results in
up to 500,000 deaths annually. 20,000 genes
• Neuraminidase is a major
glycoprotein on the influenza virus • How many might be targets for drug therapy?
membrane envelope that is
essential for viral replication and • More than 3000 experimental drugs are
infectivity. presently under study and testing
• Tamiflu is a neuraminidase
inhibitor and antiviral agent based • These and many future drugs will be designed
on the transition state of the
neuraminidase reaction. as transition-state analog inhibitors
• See the DrugBank: http://drugbank.ca
 How to read and write mechanisms
• The custom of writing chemical reaction mechanisms
with electron dots and curved arrows began with
Gilbert Newton Lewis and Sir Robert Robinson
• Learning to read and write mechanisms should begin
with a review of Lewis dot structures
• And with a review of the concepts of valence
electrons and formal charge
• Formal charge = group number – nonbonding
electrons – (½ shared electrons)
• Electronegativity is also important:
F>O>N>C>H
How to read and write mechanisms
For a bond-breaking event, the arrow begins in the middle of
the bond, and the arrow points to the atom that will accept the
electrons.
How to read and write mechanisms
• It has been estimated that 75% of the steps in enzyme
reaction mechanisms are proton (H+) transfers.
• If the proton is donated or accepted by a group on the
enzyme, it is often convenient (and traditional) to represent
the group as “B”, for “base”, even if B is protonated and
behaving as an acid:
How to read and write mechanisms
• In written mechanisms, a curved arrow shows
the movement of an electron pair
• And thus the movement of a pair of electrons
from a filled orbital to an empty one
• A full arrowhead represents an electron pair
• A half arrowhead represents a single electron
• For a bond-breaking event, the arrow begins
in the middle of the bond
How to read and write mechanisms
For a bond-making event, the arrow begins at the source of the
electrons (for example, a nonbonded pair), and the arrowhead
points to the atom where the new bond will be formed.
How to read and write mechanisms
• It is important to appreciate that a proton transfer
can change a nucleophile into an electrophile, and
vice versa.
• Thus, it is necessary to consider:
– The protonation states of substrate and active-site
residues
– How pKa values can change in the environment of
the active site
• For example, an active-site histidine, which might
normally be protonated, can be deprotonated by
another group and then act as a base, accepting a
proton from the substrate
 How to read and write mechanisms
An active-site histidine, which
might normally be protonated, can
be deprotonated by another group
and then act as a base, accepting
a proton from the substrate.
14.5 What Are the Mechanisms of Catalysis?
• Enzymes facilitate formation of near-attack
complexes
• Protein motions are essential to enzyme catalysis
• Covalent catalysis
• General acid-base catalysis
• Low-barrier hydrogen bonds
• Metal ion catalysis
Enzymes facilitate formation of near-attack
complexes
• Thomas Bruice has proposed that near-attack
conformations are precursors to transition
states
• In the absence of an enzyme, potential
reactant molecules adopt a NAC only about
0.0001% of the time
• On the other hand, NACs have been shown to
form in enzyme active sites from 1% to 70% of
the time
How to read and write mechanisms
Water can often act as an acid or base at the active site
through proton transfer with an assisting active-site residue:
This type of chemistry is the basis for general acid-base
catalysis (discussed on pages 430-431).
Enzymes facilitate formation of near-attack
complexes
• X-ray crystal structure studies and computer modeling
have shown that the reacting atoms and catalytic groups
are precisely positioned for their roles
• Such preorganization selects substrate conformations in
which the reacting atoms are in van der Waals contact and
at an angle resembling the bond to be formed in the
transition state
• Thomas Bruice has termed such arrangements near-attack
conformations (NACs)
• NACs are precursors to reaction transition states
Enzymes facilitate formation of near-attack
complexes
Figure 14.7 NACs are
characterized as having
reacting atoms within 3.2 Å
and an approach angle of
±15° of the bonding angle in
the transition state.
 Figure 14.8 The active site of liver alcohol
dehydrogenase – a near-attack complex.
Enzymes facilitate formation of near-attack complexes
Figure 14.7 In an
enzyme active site, the
NAC forms more
readily than in the
uncatalyzed reaction.
The energy separation
between the NAC and
the transition state is
approximately the
same in the presence
and absence of the
enzyme.

Protein Motions Are Essential to Enzyme Catalysis Protein Motions Are Essential to Enzyme Catalysis

• Proteins are constantly moving – bonds vibrate, side

chains bend and rotate, backbone loops wiggle and sway,
and whole domains move as a unit
• Enzymes depend on such motions to provoke and direct
catalytic events
• Protein motions support catalysis in several ways. Active
site conformation changes can:
– Assist substrate binding
– Bring catalytic groups into position
– Induce formation of NACs
Figure 14.9 Human cyclophilin A is a prolyl isomerase, which
– Assist in bond making and bond breaking catalyzes the interconversion between trans and cis
– Facilitate conversion of substrate to product conformations of proline in peptides and proteins.

Protein Motions Are Essential to Enzyme Catalysis
Protein Motions Are Essential to Enzyme Catalysis
Figure 14.10 Catalysis in enyzme active sites depends
on motion of active-site residues. Several active-site
residues undergo greater motion during catalysis than
residues elsewhere in the protein.

Figure 14.9 The active site of cyclophilin with a bound peptide

containing proline in cis and trans conformations. Motion by
active site residues promote catalysis in cyclophilin.
 Covalent Catalysis Covalent Catalysis
• Some enzymes derive much of their rate acceleration
from formation of covalent bonds between enzyme
and substrate
• The side chains of amino acids in proteins offer a
variety of nucleophilic centers for catalysis
• These groups readily attack electrophilic centers of
substrates, forming covalent enzyme-substrate
complexes
• The covalent intermediate can be attacked in a
Figure 14.11
second step by water or by a second substrate,
Examples of covalent
forming the desired product enzyme-substrate
intermediates.

Covalent Catalysis Covalent Catalysis

Figure 14.11 Examples of covalent bond formation Figure 14.11 Examples of covalent bond formation between
between enzyme and substrate. A nucleophilic center X: enzyme and substrate. A nucleophilic center X: on an
on an enzyme attacks a phosphorus atom to form a enzyme attacks a carbonyl C to form an acyl enyzme
phosphoryl enzyme intermediate. intermediate.

Covalent Catalysis Covalent Catalysis

Figure 14.11 Examples of covalent bond formation between

enzyme and substrate. A nucleophilic center X: attacks the
anomeric carbon of a glycoside, forming a glucosyl enzyme
intermediate.
 General Acid-Base Catalysis General Acid-Base Catalysis
Catalysis in which a proton is transferred in the
transition state
• "Specific" acid-base catalysis involves H+ or OH- that
diffuses into the catalytic center
• "General" acid-base catalysis involves acids and bases
other than H+ and OH-
• These other acids and bases facilitate transfer of H+ in
the transition state
• See Figure 14.12
Figure 14.12 Catalysis of p-nitrophenylacetate hydrolysis can
occur either by specific acid hydrolysis or by general base
catalysis.

Low-Barrier Hydrogen Bonds (LBHBs) Low-Barrier Hydrogen Bonds (LBHBs)

• The typical H-bond strength is 10-30 kJ/mol, and the
O-O separation is typically 0.28 nm
• As distance between heteroatoms becomes smaller
(<0.25 nm), H bonds become stronger
• Stabilization energies can approach 60 kJ/mol in
solution
• pKa values of the two electronegative atoms must be
similar
• Energy released in forming an LBHB can assist Figure 14.13 Energy diagrams for conventional H bonds (a),
catalysis and low-barrier hydrogen bonds (b and c). In (c), the O-O
distance is 0.23 to 0.24 nm, and bond order for each O-H
interaction is 0.5.

Quantum Mechanical Tunneling Quantum Mechanical Tunneling

• Tunneling provides a path “around” the usual energy
of activation for steps in chemical reactions
• Many enzymes exploit this
• According to quantum theory, there is a finite
probability that any particle can appear on the other
side of an activation barrier for a reaction step
• The likelihood of tunneling depends on the distance
over which a particle must move
• Only protons and electrons have a significant
probability of tunneling
• The de Broglie equation relates the “de Broglie
wavelength” to the mass and energy of a particle
h
λ=
2mE
• Tunneling can only play a significant role in a reaction
when the wavelength of the transferring particle is
similar to the distance over which it is transferred
• de Broglie wavelengths for protons and electrons are
0.9Å and 38Å, respectively
• Tunneling probably contributes to most, if not all,
hydrogen transfer reactions
 Tunneling between donor and acceptor
Figure 14.13d If the distance for particle transfer is
sufficiently small, overlap of probability functions
(red) permit efficient quantum mechanical tunneling
between donor (D) and acceptor (A)
How Do Active-Site Residues Interact to Support
Catalysis?
• About half of the amino acids engage directly
in catalytic effects in enzyme active sites
• Other residues may function in secondary
roles in the active site:
– Raising or lowering catalytic residue pKa values
– Orientation of catalytic residues
– Charge stabilization
– Proton transfers via hydrogen tunneling
14.5 What Can Be Learned From Typical Enzyme
Mechanisms?
First Example: the serine proteases
• Enzyme and substrate become linked in a covalent
bond at one or more points in the reaction pathway
• The formation of the covalent bond provides
chemistry that speeds the reaction
• Serine proteases also employ general acid-base
catalysis
Metal Ion Catalysis
Figure 14.14 Thermolysin is an endoprotease with a catalytic
Zn2+ ion in the active site. The Zn2+ ion stabilizes the buildup of
negative charge on the peptide carbonyl oxygen, as a glutamate
residue deprotonates water, promoting hydroxide attack on the
carbonyl carbon.
How Do Active-Site Residues Interact to Support
Catalysis?
The active site of aromatic amine
dehydrogenase, showing the
relationship of Asp128, Thr172, and
Cys171. Coupling of local motions of
these residues to vibrational states
involved in proton transfer
contributes to catalysis.
The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin, subtilisin,
plasmin, TPA
• All involve a serine in catalysis - thus the name
• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but locations of
the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
always as His57, Asp102, Ser195
• Burst kinetics yield a hint of how they work
 The Serine Proteases The Catalytic Triad of the Serine Proteases

Figure 14.16 Structure of

Figure 14.15 The chymotrypsin (white) in a
amino acid complex with eglin C (blue
sequences of ribbon structure), a target
chymotrypsinogen, substrate. His57 (red) is
trypsin, and flanked by Asp102 (gold) and
elastase. Ser195 (green). The catalytic
site is filled by a peptide
segment of eglin. Note how
close Ser195 is to the peptide
that would be cleaved in the
reaction.

Serine Protease Binding Pockets are Adapted to Particular

The Catalytic Triad of the Serine Proteases Substrates

Figure 14.18 The substrate-binding pockets of trypsin,

Figure 14.17 The catalytic triad at
the active site of chymotrypsin
(and the other serine proteases.
chymotrypsin, and elastase. Asp189 (aqua) coordinates Arg and
Lys residues of substrates in the trypsin pocket. Val216 (purple)
and Thr226 (green) make the elastase pocket shallow and able to
accommodate only small, nonbulky residues. The chymotrypsin
pocket is hydrophobic.

Serine Proteases Cleave Simple Organic Esters, such as

p-Nitrophenylacetate
Serine Proteases Display Burst Kinetics

Figure 14.20
Burst kinetics in the
chymotrypsin reaction.

Figure 14.19 Chymotrypsin cleaves simple esters, in

addition to peptide bonds. p-Nitrophenylacetate has been
used in studies of the chymotrypsin mechanism.
Serine Protease Mechanism The Serine Protease Mechanism in Detail

A mixture of covalent and general acid-base catalysis

• Asp102 functions only to orient His57
• His57 acts as a general acid and base
• Ser195 forms a covalent bond with peptide to be
cleaved
• Covalent bond formation turns a trigonal C into a
tetrahedral C
• The tetrahedral oxyanion intermediate is stabilized
by the backbone N-H groups of Gly193 and Ser195

Figure 14.21 The chymotrypsin mechanism: binding of a

model substrate.

The Serine Protease Mechanism in Detail The Serine Protease Mechanism in Detail

Figure 14.21 The chymotrypsin mechanism: His57 stabilized by

a LBHB.
Figure 14.21 The chymotrypsin mechanism: the formation
of the covalent ES complex (E-Ser195–S complex) involves
general base catalysis by His57

The Serine Protease Mechanism in Detail The Serine Protease Mechanism in Detail

Figure 14.21 The chymotrypsin mechanism: The amino

Figure 14.21 The chymotrypsin mechanism: collapse of the product departs, making room for an entering water molecule.
tetrahedral intermediate releases the first product.
 The Serine Protease Mechanism in Detail The Serine Protease Mechanism in Detail

Figure 14.21 The chymotrypsin mechanism: Collapse of the

Figure 14.21 The chymotrypsin mechanism: Nucleophilic tetrahedral intermediate cleaves the covalent intermediate,
attack by water is facilitated by His57, acting as a general base. releasing the second product.

The Serine Protease Mechanism in Detail The Serine Protease Mechanism in Detail

Figure 14.21 The chymotrypsin mechanism: Carboxyl product Figure 14.21 The chymotrypsin mechanism: At the completion
release completes the serine protease mechanism. of the reaction, the side chains of the catalytic triad are
restored to their original states.

Transition-State Stabilization in the Serine

Proteases The “oxyanion hole”

• The chymotrypsin mechanism involves two

tetrahedral oxyanion transition states
• These transition states are stabilized by a pair
of amide groups that is termed the “oxyanion
hole”
• The amide N-H groups of Ser195 and Gly193 The oxyanion hole of
provide primary stabilization of the chymotrypsin stabilizes the
tetrahedral oxyanion transition
tetrahedral oxyanion state seen in the mechanism
of Figure 14.21.
 Aspartic proteases play many roles in humans The Aspartic Proteases
Pepsin, chymosin, cathepsin D, renin and
HIV-1 protease
• All involve two Asp residues at the active site
• These two Asp residues work together as
general acid-base catalysts
• Most aspartic proteases have a tertiary
structure consisting of two lobes (N-terminal
and C-terminal) with approximate two-fold
symmetry
• HIV-1 protease is a homodimer

The Aspartic Proteases The Aspartic Proteases

Figure 14.22 Structures of (a)
HIV-1 protease, a dimer, and (b) Figure 14.23 pH-
pepsin, a monomer. Pepsin’s rate profile for
N-terminal half is shown in red; pepsin.
the C-terminal half is shown in
blue.

Most aspartic proteases exhibit a

two-lobed structure. Each lobe
contributes one catalytic aspartate
to the active site. HIV-1 protease
is a homodimeric enzyme, with
each subunit contributing a
catalytic Asp residue.

The Aspartic Proteases Aspartic Protease Mechanism

• Aspartic proteases show one relatively low pKa,
and one relatively high pKa
• This was once thought to represent pKa values of
the two aspartate residues, but this is no longer
believed to be the case
• Instead, molecular dynamics simulations show that
aspartic proteases employ low-barrier hydrogen
bonds (LBHBs) in their mechanism
• The predominant catalytic factor in aspartic
proteases is general acid-base catalysis
Figure 14.23 pH-rate profile of HIV-1 protease.
 Aspartic Proteases May Employ Hydrogen Tunneling for
A Mechanism for the Aspartic Proteases Rate Acceleration

Figure 14.24 Mechanism for the aspartic proteases. LBHBs

play a role in states E, ES, ET', EQ', and EP'Q. Figure 14.25 Energy level diagram for the aspartic protease reaction,
showing hydrogen tunneling.

HIV-1 Protease Proteolytic cleavage pattern for the HIV genome

A novel aspartic protease

• HIV-1 protease cleaves the polyprotein
products of the HIV genome
• HIV-1 protease is a remarkable imitation of
mammalian aspartic proteases
• HIV-1 protease is a homodimer - more genetically
economical for the virus
• Active site is two-fold symmetric
Figure 14.26 HIV mRNA
provides the genetic information
for synthesis of a polyprotein.
Cleavage by HIV-1 protease
yields the active products.

Protease Inhibitors Block the Active Site of HIV-1

Protease Inhibitors Give Life to AIDS Patients
Protease
Protease inhibitors as AIDS drugs
• If HIV-1 protease can be selectively inhibited, then
new HIV particles cannot form
• Several novel protease inhibitors are currently
marketed as AIDS drugs
• Many such inhibitors work in a culture dish
• However, a successful drug must be able to kill the
virus in a human subject without blocking other
Figure 14.27 HIV-1 protease complexed with the inhibitor
essential proteases in the body
Crixivan (red) made by Merck. The “flaps” that cover the
active site are green; the catalytic active-site Asp residues
are violet.
 Chorismate Mutase: A Model for Understanding
Protease Inhibitors Give Life to AIDS Patients Catalytic Power and Efficiency
• Direct comparison of enzyme-catalyzed reactions and their
uncatalyzed counterparts is difficult
• Chorismate mutase has become a model for making this
comparison, thanks to the efforts of a large number of
enzyme mechanism researchers
• Chorismate mutase acts in the biosynthesis of
phenylalanine and tyrosine in microorganisms and plants
• It involves a single substrate and catalyzes a concerted
intramolecular rearrangement of chorismate to
prephenate
• One C-O bond is broken and one C-C bond is formed
Protease inhibitor drugs used by AIDS patients

Chorismate Mutase: A Model for Understanding Chorismate Mutase: A Model for Understanding
Catalytic Power and Efficiency Catalytic Power and Efficiency

Figure 14.28 A classic Claisen rearrangement. Conversion of

Figure 14.28 The chorismate mutase reaction converts allyl phenyl ether to 2-allyl alcohol proceeds through a
chorismate to prephenate in an intramolecular rearrangement. cyclohexadienone intermediate, which then undergoes a keto-
enol tautomerization.

The chorismate mutase reaction (and its uncatalyzed A transition-state analog for the chair mechanism
counterpart) occur via chair states of chorismate mutase
Figure 14.29 Jeremy Knowles has shown that both
The critical H the chorismate mutase and its
atoms are uncatalyzed solution counterpart
distinguished proceed via a chair conformation. A
in this figure transition-state analog of this
by blue and conformation has been characterized.
green colors.
 Transition state stabilization by electrostatic and
The structure of E. coli chorismate mutase hydrogen-bonding interactions
Figure 24.30 (a) the chorismate mutase homodimer

Figure 14.31

Twelve
electrostatic and
hydrogen-
bonding
interactions
stabilize the
transition-state
analog.

(b) The active site, showing the bound transition-state analog.

The Chorismate Mutase Active Site Favors a Near-

The Chorismate Mutase Mechanism
Attack Conformation
Figure 14.32
The Figure 14.33 Chorismate
carboxyvinyl bound to the active site of
group folds up chorismate mutase in a
and over the structure that resembles a
chorismate ring near-attack complex.
and the Arrows indicate
reaction hydrophobic interactions
proceeds via an and red dotted lines
internal indicate electrostatic
rearrangement. interactions.

Formation of a NAC is facile in the chorismate

mutase active site
Figure 14.34
Chorismate
mutase facilitates
NAC formation.
The energy
required to move
from the NAC to
the transition Chapter 15
state is essentially Enzyme Regulation
equivalent in the
catalyzed and
uncatalyzed
reactions.
15.1 – What Factors Influence Enzymatic Activity? 15.1 – What Factors Influence Enzymatic Activity?

• The availability of substrates and cofactors usually determines how

fast the reaction goes
• As product accumulates, the apparent rate of the enzymatic
reaction will decrease
• Genetic regulation of enzyme synthesis and decay determines the
amount of enzyme present at any moment
• Enzyme activity can be regulated allosterically
• Enzyme activity can be regulated through covalent modification
• Zymogens, isozymes, and modulator proteins may play a role Figure 15.1 Enzyme regulation by reversible covalent
modification.

Figure 15.3 The proteolytic activation of

15.1 – What Factors Influence Enzymatic Activity?
chymotrypsinogen

Zymogens are inactive precursors

of enzymes. Typically, proteolytic
cleavage produces the active
enzyme.

Figure 15.2 Proinsulin is an 86-

residue precursor to insulin

Proteolytic Enzymes of the Digestive Tract The Cascade of Activation Steps Leading to Blood Clotting

Figure 15.4 The intrinsic

and extrinsic pathways
converge at factor X, and
the final common pathway
involves the activation of
thrombin and its
conversion of fibrinogen
into fibrin, which
aggregates into ordered
filamentous arrays that
crosslink to form the clot.
 Isozymes Are Enzymes With Slightly Different 15.2 – What Are the General Features of Allosteric
Subunits Regulation?

Action at "another site"

• Enzymes situated at key steps in metabolic pathways
are modulated by allosteric effectors
• These effectors are usually produced elsewhere in the
pathway
• Effectors may be feed-forward activators or feedback
inhibitors
• Kinetics are sigmoid ("S-shaped")

Figure 15.5 The

isozymes of lactate
dehydrogenase (LDH).

15.2 – What Are the General Features of Allosteric 15.3 Can Allosteric Regulation Be Explained by
Regulation? Conformational Changes in Proteins?

Monod, Wyman, Changeux (MWC) Model:

- Allosteric proteins can exist in two states: R (relaxed)

and T (taut)
- In this model, all the subunits of an oligomer must be
in the same state
- T state predominates in the absence of substrate S
- S binds much tighter to R than to T

Figure 15.6 Sigmoid v versus [S] plot. The dotted line represents
the hyperbolic plot characteristic of normal Michaelis=Menten
kinetics.

The Symmetry Model for Allosteric Regulation is Based on The Symmetry Model for Allosteric Regulation is Based
Two Conformational States for a Protein on Two Conformational States for a Protein

Figure 15.7 Allosteric effects: A and I binding to R and T,

Figure 15.7 Allosteric effects: A and I binding to R and T, respectively.
respectively.
The Symmetry Model for Allosteric Regulation is Based
on Two Conformational States for a Protein
Figure 15.7 Allosteric effects: A and I
binding to R and T, respectively.
The Sequential Model for Allosteric Regulation is Based
on Ligand-Induced Conformation Changes
• An alternative model – proposed by Koshland,
Nemethy, and Filmer (the KNF model) relies on the
idea that ligand binding triggers a conformation
change in a protein
• If the protein is oligomeric, ligand-induced
conformation changes in one subunit may lead to
conformation changes in adjacent subunits
• The KNF model explains how ligand-induced
conformation changes could cause subunits to adopt
conformations with little affinity for the ligand – i.e.,
negative cooperativity
• The KNF model is termed the sequential model
The Sequential Model for Allosteric Regulation is Based
on Ligand-Induced Conformation Changes
Figure 15.8 The
Koshland-Nemethy-
Filmer model.
Theoretical curves
for the binding of a
ligand to a protein
having four identical
subunits, each with
one binding site for
the ligand.
More about the MWC model
• Cooperativity is achieved because S binding
increases the population of R, which increases
the sites available to S
• Ligands such as S are positive homotropic
effectors
• Molecules that influence the binding of
something other than themselves are
heterotropic effectors
The Sequential Model for Allosteric Regulation is Based
on Ligand-Induced Conformation Changes
Figure 15.8 The Koshland-Nemethy-
Filmer sequential model for allosteric
behavior. (a) S binding can, by induced
fit, cause a conformational change in the
subunit to which it binds. (b) If subunit
interactions are tightly coupled, binding of
S to one subunit may cause the other
subunit to assume a conformation having
a greater or lesser affinity for S. That is,
the ligand-induced conformational
change in one subunit can affect the
adjoining subunit.
The notable difference between MWC and KNF models
• In the MWC model, the different
conformations have different affinities for the
various ligands, and the concept of ligand-
induced conformational changes is ignored
• In contrast, the KNF model is based on ligand-
induced conformational changes
 15.4 What Kinds of Covalent Modification Regulate 15.4 What Kinds of Covalent Modification Regulate
the Activity of Enzymes? the Activity of Enzymes?

• Enzyme activity can be regulated through reversible
phosphorylation
• This is the most prominent form of covalent
modification in cellular regulation
• Phosphorylation is accomplished by protein kinases
• Each protein kinase targets specific proteins for
phosphorylation
• Phosphoprotein phosphatases catalyze the reverse
reaction – removing phosphoryl groups from
proteins
• Kinases and phosphatases themselves are targets of
regulation
• Protein kinases phosphorylate Ser, Thr, and Tyr
residues in target proteins
• Kinases typically recognize specific amino acid
sequences in their targets
• In spite of this specificity, all kinases share a common
catalytic mechanism based on a conserved core
kinase domain of about 260 residues (see Figure
15.9)
• Kinases are often regulated by intrasteric control, in
which a regulatory subunit (or domain) has a
pseudosubstrate sequence that mimics the target
sequence, minus the phosphorylatable residue

15.4 What Kinds of Covalent Modification Regulate 15.4 What Kinds of Covalent Modification Regulate
the Activity of Enzymes? the Activity of Enzymes?

Figure 15.9 Protein kinase A is

shown complexes with a
pseudosubstrate peptide
(orange).

This complex also includes ATP

(red) and two Mn2+ ions (yellow)
bound at the active site.

Cyclic AMP-dependent protein kinase is composed of Phosphorylation is Not the Only Form of Covalent
catalytic and regulatory subunits Modification that Regulates Protein Function

• Several hundred different chemical modifications of

proteins have been discovered
• Only a few of these are used to achieve metabolic
regulation through reversible conversion of an
enzyme between active and inactive forms
• A few are summarized in Table 15.3
Figure 15.10 cyclic AMP-dependent protein kinase (also
• Three of the modifications in Table 15.3 require
known as protein kinase A (PKA) is a 150- to 170-kD R2C2
tetramer in mammalian cells. nucleoside triphosphates (ATP, UTP) that are related
to cellular energy status
The two R (regulatory) subunits bind cAMP; cAMP binding
releases the R subunits from the C (catalytic) subunits. C
subunits are enzymatically active as monomers.
 Phosphorylation is Not the Only Form of Covalent
Modification that Regulates Protein Function
Acetylation in Enzyme Regulation

• Acetylation is a prominent modification for the

regulation of metabolic enzymes
• Acetylation of an ε-NH3+ group on a Lys residue
changes it from a positively charged amino group to
a neutral amide
• This change may have consequences for protein
structure and thus function
• The acetylating enzyme is termed an acetyl-CoA-
dependent lysine acetyltransferase or KAT
• More than 30 KATs are known in mammals
• Deacetylation by KDACs (lysine deacetylases)
reverse the effects of acetylation

Acetylation in Enzyme Regulation Acetylation in Enzyme Regulation

• Proteomics studies show that acetylation of metabolic

enzymes is an important mechanism for regulating the flow of
metabolic substrates (carbohydrates and fats, for example)
through the central metabolic pathways
• Acetylation activates some enzymes and inhibits others
• Cellular levels of major metabolic fuels such as glucose, fatty
acids, and amino acids influence the degree of acetylation
• The KDACs include sirtuins, a class of NAD+-dependent
protein deacetylating enzymes
• Sirtuins are implicated in energy metabolism and longevity
Figure 15.11 Activation of malate dehydrogenase
by acetylation

15.5 Are Some Enzymes Controlled by Both Allosteric GP converts glycogen into readily usable fuel in the
Regulation and Covalent Modification? form of glucose-1-phosphate

• Glycogen phosphorylase (GP) is an example of the

many enzymes that are regulated both by allosteric
controls and by covalent modification
• GP cleaves glucose units from nonreducing ends of
glycogen
• This converts glycogen into readily usable fuel in the
form of glucose-1-phosphate
• This is a phosphorolysis reaction
• Muscle GP is a dimer of identical subunits, each with
PLP covalently linked Figure 15.12 The glycogen phosphorylase reaction
• There is an allosteric effector site at the subunit
interface
 Phosphoglucomutase converts glucose-1-P into the
glycolytic substrate, glucose-6-P
Figure 15.13 The phosphoglucomutase reaction.
The structure of glycogen phosphorylase
Figure 15.14 The structure of
glycogen phosphorylase.
Glycogen Phosphorylase Activity is Regulated
Allosterically
Figure 15.15 v versus S curves for glycogen phosphorylase.
(a)The response to the concentration of the substrate
phosphate (Pi).
(b)ATP is a feedback inhibitor.
(c)AMP is a positive effector. It binds at the same site as ATP.
The structure of glycogen phosphorylase
• Glycogen phosphorylase is a dimer of identical 842
residue subunits
• Each subunit contains an active site (at the center of
the subunit) and an allosteric effector site near the
subunit interface
• A regulatory phosphorylation site is located at Ser14
on each subunit
• A glycogen-binding site exerts regulatory control
• Each subunit contributes a “tower helix” (residues
262 to 278) to the subunit-subunit interface
• In the dimer, the tower helices extend from their
respective subunits and pack against each other
Glycogen Phosphorylase Activity is Regulated
Allosterically
• Muscle glycogen phosphorylase shows cooperativity in
substrate binding
• ATP and glucose-6-P are allosteric inhibitors of glycogen
phosphorylase
• AMP is an allosteric activator of glycogen phosphorylase
• When ATP and glucose-6-P are abundant, glycogen
breakdown is inhibited
• When cellular energy reserves are low (i.e., high [AMP]
and low [ATP] and [G-6-P]) glycogen catabolism is
stimulated
Glycogen phosphorylase conforms to the MWC
model
• The active form of the enzyme is designated the R
state
• The inactive form of the enzyme is denoted the T
state
• AMP promotes the conversion to the active state
• ATP, glucose-6-P, and caffeine favor conversion to the
inactive T state
• A significant conformation change occurs at the
subunit interface between the T and R state
• This conformational change at the interface is linked
to a structural change at the active site that affects
catalysis
 Glycogen Phosphorylase is Controlled by Both A Conformation Change Regulates Activity of
Allosteric Regulation and Covalent Modification Glycogen Phosphorylase
Figure 15.17 The major
conformational change that
occurs in the N-terminal
Figure 15.16 The mechanism
residues upon phosphorylation
of covalent modification and
of Ser14. Ser14 is shown in red.
allosteric regulation of
glycogen phosphorylase.
N-terminal conformation of
phosphorylated enzyme
(phosphorylase a): yellow.

N-terminal conformation of
unphosphorylated enzyme
(phosphorylase b): cyan.

Regulation of GP by Covalent Glycogen phosphoryase is activated by a

Modification cascade of reactions
• In 1956, Edwin Krebs and Edmond Fischer
showed that a ‘converting enzyme’ could
convert phosphorylase b to phosphorylase a
• Three years later, Krebs and Fischer showed that
this conversion involves covalent
phosphorylation
• This phosphorylation is mediated by an enzyme
cascade (Figure 15.18)

Figure 15.18 The hormone-activated enzymatic cascade

that leads to activation of glycogen phosphorylase.

The Adenylyl Cyclase Reaction The Adenylyl Cyclase Reaction

(Shown here are the

products of the reaction.
ATP, the reactant, is
shown on the previous
slide.)

Figure 15.19 The adenylyl cyclase reaction. The reaction is

driven forward by subsequent hydrolysis of pyrophosphate by
the enzyme inorganic pyrophosphatase.
Figure 15.19 The adenylyl cyclase reaction. The reaction is
driven forward by subsequent hydrolysis of pyrophosphate by
the enzyme inorganic pyrophosphatase.
 cAMP is a Second Messenger cAMP is a Second Messenger

• Cyclic AMP is the intracellular agent of

extracellular hormones - thus a ‘second
messenger’
• Hormone binding stimulates a GTP-binding
protein (G protein), releasing Gα(GTP)
• Binding of Gα(GTP) stimulates adenylyl cyclase to
make cAMP
Figure 15.20 Hormone binding to its
receptor leads via G-protein
activation to cAMP synthesis.
Adenylyl cyclase and the hormone
receptor are integral membrane
proteins; Gα and Gβγ are membrane-
anchored proteins.

Figure 15.21 O2-binding curves for hemoglobin and

Hemoglobin myoglobin
A classic example of allostery
• Hemoglobin and myoglobin are oxygen- transport
and oxygen-storage proteins, respectively
• Compare the oxygen-binding curves for
hemoglobin and myoglobin
• Myoglobin is monomeric; hemoglobin is tetrameric
• Mb: 153 aa, 17,200 MW
• Hb: two α chains of 141 residues, 2 β chains of 146
residues

The structure of myoglobin is similar to that of

an Hb monomer Mb and Hb use heme to bind Fe2+

Figure 15.22 The myoglobin and

hemoglobin structures.

Myoglobin is monomeric

Hemoglobin is tetrameric

Figure 15.23 Heme is formed when protoporphyrin IX binds Fe2+

 Fe2+ is coordinated by His F8 Fe2+ is coordinated by His F8
• Iron interacts with six ligands in Hb and Mb
• Four of these are the N atoms of the porphyrin
• A fifth ligand is donated by the imidazole side chain
of amino acid residue His F8
• (This residue is on the sixth or “F” helix, and it is the
8th residue in the helix, thus the name.)
• When Mb or Hb bind oxygen, the O2 molecule adds
to the heme iron as the sixth ligand
• The O2 molecule is tilted relative to a perpendicular
to the heme plane
Figure 15.24 The six
liganding positions of an
iron atom in Hb and Mb.

Myoglobin Structure O2 Binding Alters Mb Conformation

• Mb is a monomeric heme protein
• Mb polypeptide "cradles" the heme group
• Fe in Mb is Fe2+ - ferrous iron - the form that binds
oxygen
• Oxidation of Fe yields 3+ charge - ferric iron
• Mb with Fe3+ is called metmyoglobin and does not
bind oxygen
• In deoxymyoglobin, the ferrous ion actually lies 0.055
nm above the plane of the heme
• When oxygen binds to Fe in heme of Mb, the heme Fe
is drawn toward the plane of the porphyrin ring
• With oxygen bound, the Fe2+ atom is only 0.026 nm
above the plane
• For Mb, this small change has little consequence
• But a similar change in Hb initiates a series of
conformational changes that are transmitted to
adjacent subunits

Cooperative Binding of Oxygen Influences

Hb Has an α2β2 Tetrameric Structure Hemoglobin Function

• Mb, an oxygen-storage protein, has a greater affinity

for oxygen at all oxygen pressures
Figure 15.25 An αβ
dimer of Hb, with
• Hb is different – it must bind oxygen in lungs and
packing contacts release it in capillaries
indicated in blue. • Hb becomes saturated with O2 in the lungs, where the
The sliding contacts
partial pressure of O2 is about 100 torr
made with the other • In capillaries, pO2 is about 40 torr, and oxygen is
dimer are shown in released from Hb
yellow. The changes in
these sliding contacts • The binding of O2 to Hb is cooperative – binding of
are shown in Figure oxygen to the first subunit makes binding to the other
15.26. subunits more favorable
 O2-Binding Curves of Mb and Hb
The oxygen binding curve of
Mb resembles an
enzyme:substrate saturation
curve.
An Alternative O2-Binding Curve for Hb
Oxygen saturation
curve for Hb in
the form of Y
versus pO2
assuming n=4
and P50 =26 torr.
Y is the fractional
saturation of Hb:

[ pO2 ]4
Y=
[ pO2 ]4 + K

An Alternative O2-Binding Curve for Hb The Conformation Change

The secret of Mb and Hb
A comparison of • Oxygen binding changes the Mb conformation
the experimentally
observed O2 curve
• Without oxygen bound, Fe2+ is out of heme plane
for Hb yielding a • Oxygen binding pulls the Fe2+ into the heme plane
value for n of 2.8, • Fe2+ pulls its His F8 ligand along with it
the hypothetical
curve if n=4, and
• The F helix moves when oxygen binds
the curve if n=1 • Total movement of Fe2+ is 0.029 nm – i.e., 0.29 Å
(non-interacting • This change means little to Mb, but lots to Hb!
O2-binding sites).

Oxygen Binding by Hb Induces a Quaternary Oxygen binding to Hb results in a 15° rotation of one
Structure Change αβ pair relative to the other

• When deoxy-Hb crystals are exposed to oxygen, they

shatter. Evidence of a large-scale structural change
• One alpha-beta pair moves relative to the other by 15
degrees upon oxygen binding
• This massive change is induced by movement of Fe by
0.039 nm when oxygen binds
Figure 15.26 Subunit motion in hemoglobin when the
molecule goes from the (a) deoxy form to the (b) oxy form.
 Fe2+ Movement by Less Than 0.04 nm Induces the Fe2+ Movement by Less Than 0.04 nm Induces the
Conformation Change in Hb Conformation Change in Hb
• In deoxy-Hb, the iron atom lies out of the heme plane by
about 0.06 nm Figure 15.27
• Upon O2 binding, the Fe2+ atom moves about 0.039 nm Changes in the
closer to the plane of the heme position of the heme
iron atom upon
• It is as if the O2 is drawing the heme iron into the plane oxygenation lead to
• This may seem like a trivial change, but its biological conformational
consequences are far-reaching changes in the
hemoglobin
• As Fe2+ moves, it drags His F8 and the F helix with it molecule.
• This change is transmitted to the subunit interfaces,
where conformation changes lead to the rupture of salt
bridges

The Physiological Significance of the Hb:O2

Salt bridges that stabilize deoxy-Hb are broken in oxy- Interaction
Hb
• Hb must be able to bind oxygen in the lungs
Figure 15.28 Salt bridges between • Hb must be able to release oxygen in capillaries
different subunits in human deoxy-Hb.
• If Hb behaved like Mb, very little oxygen would be
These noncovalent, electrostatic
interactions are disrupted upon
released in capillaries - see Figure 15.21!
oxygenation. • The sigmoid, cooperative oxygen-binding curve of Hb
(a)The salt bridges and H-bonds makes its physiological actions possible!
involving interactions between N-
terminal and C-terminal residues in the
α-chains.
(b)The salt bridges and H bonds
involving C-terminal residues of β-
chains

H+ Promotes Dissociation of Oxygen from Hemoglobin H+ Promotes Dissociation of Oxygen from Hemoglobin

• Binding of O2 to Hb is affected by several

agents, including H+, CO2, and chloride ions
• The effect of H+ is particularly important Figure 15.29 The
oxygen saturation
• Deoxy-Hb has a higher affinity for H+ than oxy- curves for myoglobin
Hb and for hemoglobin at
five different pH
• Thus, as pH decreases, dissociation of O2 from values: 7.6, 7.4,7.2,
hemoglobin is enhanced 7.0, 6.8.
• Ignoring the stoichiometry of O2 and H+, we
can write:
HbO2 + H+
HbH+ + CO2

The Antagonism of O2 Binding by H+ is Termed
the Bohr Effect
• The effect of H+ on O2 binding was discovered by
Christian Bohr (the father of Neils Bohr, the
atomic physicist)
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance
CO2 Also Promotes the Dissociation of O2 from
Hemoglobin
Figure 15.30 Oxygen binding curves of
blood and of hemoglobin in the
absence and presence of CO2 and
BPG.
2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
• In the absence of 2,3-BPG, oxygen binding to Hb
follows a rectangular hyperbola!
• The sigmoid binding curve is only observed in the
presence of 2,3-BPG
• Since 2,3-BPG binds at a site distant from the Fe
where oxygen binds, it is called an allosteric
effector
CO2 Also Promotes the Dissociation of O2 from
Hemoglobin
Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues and extremities leads
to proton production:
CO2 + H2O
H+ + HCO3–
• These protons are taken up by Hb as oxygen
dissociates
• The reverse occurs in the lungs
Summary of the Physiological Effects of H+ and CO2 on
O2 Binding by Hemoglobin
• At the tissue-capillary interface, CO2 hydration and
glycolysis produce extra H+, promoting additional
dissociation of O2 where it is needed most
• At the lung-artery interface, bicarbonate dehydration
(required for CO2 exhalation) consumes extra H+,
promoting CO2 release and O2 binding
BPG Binding to Hb Has Important Physiological
Significance
Figure 15.31 The structure, in ionic form of BPG or 2,3-
bisphosphoglycerate, an important allosteric effector of Hb
 BPG Binding to Hb Has Important Physiological BPG Binding to Hb Has Important Physiological
Significance Significance
The "inside" story......
• Where does 2,3-BPG bind?
– "Inside"
– in the central cavity
• What is special about 2,3-BPG?
– Negative charges interact with 8 positive charges
in the cavity: 2 Lys, 4 His, 2 N-termini
• Fetal Hb - lower affinity for 2,3-BPG, higher affinity
for oxygen, so it can get oxygen from mother
Figure 15.32 The ionic binding of
BPG to the two β-subunits of Hb.
BPG lies at the center of the cavity
between the two β-subunits.

Fetal Hemoglobin Has a Higher Affinity for O2 Because Fetal Hemoglobin Has a Higher Affinity for O2 Because
it has a Lower Affinity for BPG it has a Lower Affinity for BPG
• The fetus depends on its mother for O2, but its
circulatory system is entirely independent
• Gas exchange takes place across the placenta
• Fetal Hb differs from adult Hb – with γ-chains in
place of β-chains – and thus a α2γ2 structure
• As a result, fetal Hb has a higher affinity for O2
• Why does fetal Hb bind O2 more tightly?
Figure 15.33 Comparison of the
• Fetal γ-chains have Ser instead of His at position 143 oxygen saturation curves of Hb A and
and thus lack two of the positive charges in the BPG- Hb F under similar conditions of pH
binding cavity and [BPG].
• BPG binds less tightly and Hb F thus looks more like
Mb in its O2 binding behavior

Sickle-Cell Anemia is a Molecular Disease Sickle-Cell Anemia is a Molecular Disease

• Sickle-cell anemia patients have abnormally-shaped Figure 15.34 The polymerization of Hb S molecules arises
red blood cells because Val replaces His on the surface of β-chains. The
• The erythrocytes are crescent-shaped instead of disc- “block” extending from Hb S below represents the Val side
chains. These can insert into hydrophobic pockets in
shaped
neighboring Hb S molecules.
• The sickle cells pass less freely through the
capillaries, impairing circulation and causing tissue
damage
• A single amino acid substitution in the β-chains of Hb
causes sickle-cell anemia
• Glu at position 6 of the β-chains is replaced by Val
• As a result, Hb S molecules aggregate into long,
chainlike polymeric structures
 Sickle-Cell Anemia is a Molecular Disease Sickle-Cell Anemia is a Molecular Disease

Figure 15.34 A dimer of

Hb S tetramers. Val at
position 6 of the β-chains
is shown in blue. Hemes
are red.

Figure 15.34 Polymerization of HB S.

Sickle-Cell Anemia is a Molecular Disease

Hemoglobin and Nitric Oxide
Figure 15.34 Structure of the polymerized Hb S filament. Val
• Nitric oxide (NO·) is a simple gaseous molecule that
at position 6 of the β-chains is shown in blue. Hemes are red.
acts as a neurotransmitter and as a second
messenger in signal transduction (see Chapter 32)
• NO· is a high-affinity ligand for Hb, binding to the
heme iron 10,000 times more tightly than O2
• So why is NO· not bound instantaneously to Hb,
preventing its physiological effects?
• NO· reacts with the –SH of Cys93β, forming an S-
nitroso derivative:

Hemoglobin and Nitric Oxide

• The S-nitroso group is in equilibrium with other S-
nitroso compounds formed by reaction of nitric oxide
with small-molecule thiols such as free Cys or
glutathione:

• These small-molecule thiols transfer NO· from

erythrocytes to endothelial receptors, where it exerts
its physiological effects