Enzymes: Kinetics,

Specificity, and
Regulation

Outline
Enzymes Catalytic Power, Specificity,

and Regulation
Introduction to Enzyme Kinetics
Kinetics of Enzyme-Catalyzed Reactions
Enzyme Inhibition
Kinetics of Enzyme-Catalyzed Reactions
Involving >1 Substrates
RNA and Antibody Molecules As
Enzymes: Ribozymes & Abzymes
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Outline contd
Specificity Is the Result of Molecular

Recognition
Controls over Enzymatic Activity
The Allosteric Regulation of Enzyme
Activity
A Model for the Allosteric Behavior of
Proteins
A Paradigm of Enzyme Regulation:
Glycogen Phosphorylase
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Enzymes
Enzymes endow cells with the remarkable

capacity to exert kinetic control over

thermodynamic potentiality
Enzymes are the agents of metabolic

function

Enzymes
Biological catalysts
that function in dilute, aqueous solutions under
mild cellular conditions (e.g., pH and
temperature) to increase reaction rate
Catalytic power
Ratio of catalyzed rate to uncatalyzed rate
Specificity
Regulation of catalysis
Enzyme levels and types regulated genetically
Inhibitors and activators modify enzyme activity
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Types of Enzymes
Simple enzymes

Composed solely of protein, single or multiple

subunits
Complex enzymes

Protein plus small organic molecule(s) or metal

Entire complex called holoenzyme
Protein part called apoenzyme
Non-protein part called coenzyme or
prosthetic group
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Non-protein Components
Cofactors
Inorganic molecules
Coenzymes
Organic molecules
Often vitamins

Prosthetic group
Firmly bound coenzyme
Holoenzyme
Apoenzyme plus prosthetic group
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Catalytic Power
Enzymes can accelerate reactions as much

as 1016 over uncatalyzed rates!

Urease is a good example:
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10

-10/sec

Ratio is 1x1014 !

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Specificity
Enzymes selectively recognize proper

substrates over other molecules

Enzymes produce products in very high
yields - often much greater than 95%
Specificity is controlled by structure - the
unique fit of substrate with enzyme
controls the selectivity for substrate and
the product yield
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Other Aspects of Enzymes

Regulation - to be covered in Chapter 14

Mechanisms - to be covered in Chapter 11
Coenzymes - to be covered in Chapter 22

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Enzyme Kinetics
Several terms to know!
rate or velocity
rate constant
rate law
order of a reaction
molecularity of a reaction

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Kinetics
Kinetics is the study of the rate of

change of reactants to products

Velocity refers to the change in conc. Of
substrate or product per unit time
Rate refers to the change in total quantity
per unit time
Initial velocity is the change in reactant
or product conc. during the linear phase of
a reaction
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Chemical Kinetics
Rate law

A
P

dA

k [ A] n

dt
k = rate constant
n = order of reaction

Molecularity
Number of molecules that must simultaneously react
Unimolecular
n = 1 k = first order rate constant
Bimolecular
k second order rate constant
n = 2, or
v = k[A][B]

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Reaction rates
Limited by activation barrier
Free energy needed to reach transition state
Arrhenius equation

G tr
RT

k Ae

Reaction rate is influenced by

Temperature
Catalysts

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Chemical Reactions and Rates

For reaction: AB the rate can be

expressed as either the decrease in conc.

of A or the increase in conc. of B
k[B] (neg. sign indicates decrease)
[B] = k[A]
-[A] =

The k is the rate constant which is

related to the equilibrium constant, Keq

Keq refers to the state where the forward
and reverse reactions are equal
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Chemical Reactions and Rates 2

The rate constant for the forward reaction

is defined as k+1 and that of the reverse

reaction as k-1
Rate is defined as velocity (v) thus at
equilibrium vforward = vreverse
Where vforward = k+1[A] and visa versa
Therefore, Keq = [B]/[A]

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Enzyme kinetics
Saturation: Zero-order kinetics at high [S]

Michealis Menten

k1
k2

P v Vmax [S]
E S
ES
E

k 1
Km[S]

Conditions applicable
[S] initial > [Enzyme]
pH, temperature, ionic
strength,[enzyme] constant
Initial rate measured
[S] essentially equal to [So]
[P] essentially zero

K kkk
1

K m [S ]
when
V
v max
2
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Enzymes as Catalysts
Enzymes increase the rate of reactions

without themselves being altered in the

process of substrate conversion to product
This defines a catalyst
Enzymes increase reaction rates by lowering
the energy input needed to form a complex of
reactant competent to form product
This occurs via the formation of a complex
between enzyme and substrate (ES)
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Michaelis Michaelis-Menten Menten

Kinetics
Developed the rate equation used by all

biochemists
Three basic assumptions

ES complex is in a steady state, i.e. remains

constant during the initial phase of a reaction

when enzyme is saturating all is in ES
complex
if all enzyme in ES then rate of product
formation is maximal
i.e. Vmax =

k2[ES]
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Michaelis Michaelis-Menten Menten

Kinetics
The Michaelis-Menten equation is a

quantitative description of the relationship

between the rate of an enzyme catalyzed
reaction (v1), substrate concentration [S], the
M-M rate constant (Km) and maximal velocity
(Vmax)

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Michaelis Michaelis-Menten Menten

Kinetics
Utilizing the M-M equation it can be shown

that the Km is equal to the concentration of

substrate required to attain half maximal
velocity for any given reaction

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The Transition State

Understand the difference between G and
G
The overall free energy change for a

reaction is related to the equilibrium

constant
The free energy of activation for a reaction
is related to the rate constant
It is extremely important to appreciate
this distinction!
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G= G

- GS

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What Enzymes Do....

Enzymes accelerate reactions by lowering

the free energy of activation

Enzymes do this by binding the

transition state of the reaction better

than the substrate
Much more of this in Chapter 11 next

lecture!
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The Michaelis-Menten
Equation
You should be able to derive this!
Louis Michaelis and Maude Menten's theory
It assumes the formation of an enzyme-

substrate complex
It assumes that the ES complex is in rapid
equilibrium with free enzyme
Breakdown of ES to form products is assumed
to be slower than:
formation of ES and
breakdown of ES to re-form E and S
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Understanding Km

The "kinetic activator constant"

Km is a constant

Km is a constant derived from rate

constants
Km is, under true Michaelis-Menten
conditions, an estimate of the
dissociation constant of E from S
Small Km means tight binding high
affinity; high Km means weak binding
low affinity
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Understanding Vmax
The theoretical maximal velocity
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

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The dual nature of the MichaelisMenten equation

Combination of 0-order and

1st-order kinetics
When S is low, the
equation for rate is 1st
order in S
When S is high, the
equation for rate is 0order in S
The Michaelis-Menten
equation describes a
rectangular
hyperbolic
dependence of v on S!

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The Turnover Mumber

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

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The catalytic efficiency

Name for kcat/Km

An estimate of "how perfect" the enzyme

is
kcat/Km is an apparent second-order rate
constant
It measures how 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

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Enzyme parameters
International unit
Amount to catalyze formation of one
micromole of product in one minute

Turnover number
kcat = k2 = Vmax/[ET]
k1 sets upper limit on catalytic efficiency
Reaction can go no faster than rate at which E and S
form ES

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Linear Plots of the MichaelisMenten Equation

Be able to derive these equations!
Lineweaver-Burk
Hanes-Woolf
Hanes-Woolf is best - why?
Smaller and more consistent errors across

the plot
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Lineweaver Lineweaver-Burk Analysis

In practice determination of Km from

curvilinear plots is not accurate

Lineweaver and Burk manipulated the MM

equation by taking its reciprocal values

generating a double reciprocal plot

Leads to a linear graph of the reciprocals

of velocity and substrate concentration

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Plots
Direct plot
v versus [S]
v = (Vmax [S])/(Km+[S])
Rectangular hyperbola
Asymptotically approaches Vmax when

[S] high

Km = [S] when v = Vmax/2

Lineweaver-Burk
(double-reciprocal) - Linear
1/v versus 1/[S]
1/v = (Km/Vmax)(1/[S])+1/Vm
Slope = Km/Vmax
y-intercept =1/Vmax
x-intercept = -1/Km
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Temperature dependence of
enzyme-catalyzed reactions
Below 50C
Q10: Ratio of activities at

two temperatures 10
apart: For typical enzyme
Q10=2

Above 50C
Typically enzyme

denatures

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pH dependence of enzyme-catalyzed
reactions

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Enzyme Inhibitors
Reversible versus Irreversible
Reversible inhibitors interact with an

enzyme via noncovalent associations

Irreversible inhibitors interact with an

enzyme via covalent associations

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Classes of Inhibition
Two real, one hypothetical
Competitive inhibition
Noncompetitive inhibition
Uncompetitive inhibition

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Competitive inhibition
Inhibitor binds at substrate site of E
inhibitor (I) binds only to E, not to ES

substrate competes for inhibitor,

Vmax unchanged, Km increased
inhibition is reversible

as higher

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Noncompetitive inhibition
Inhibitor binds at site other than substrate,
inhibitor (I) binds either to E and/or to ES
ESI cannot form product, increased substrate does not

compete,
Km unchanged, Vmax decreased

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Uncompetitive inhibition
Inhibitor only binds to ES complex

due to binding site becoming available

only when substrate is bound,
inhibitor (I) binds only to ES, not to
E. This is a hypothetical case that
has never been documented for a
real enzyme, but which makes a
useful contrast to competitive
inhibition
Km and Vmax decreased

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Bisubstrate reactions:

E + S1 + S2 S1ES2 P1EP2 E + P1 + P2

Sequential or single displacement

Random: Either substrate binds to enzyme, either product
is released
Ordered: Leading substrate binds first followed by second
substrate

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Bisubstrate reactions:

E + S1 + S2 S1ES2 P1EP2 E + P1 + P2
Ping-Pong or double-displacement:

Leading substrate binds, Enzyme modified, Product released

Second substrate binds: Enzyme unmodified: Second product

released

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Ribozymes and Abzymes

Relatively new discoveries
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 great recent review, see Science, Vol.
269, pages 1835-1842 (1995)
We'll say more about transition states in Ch 11

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Regulation of Enzyme Activity

Control the expression of genes and get more or

less enzyme

Enzyme exists in inactive form (zymogen) that

must be modified, primarily by cleavage

Covalent modification to increase or decrease

activity, most common is phosphorylation

Sequestration
Allosteric regulation, both positive and negative
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Enzyme regulation
Approach to equilibrium

in vivo substrate
concentrations
Genetic controls
Covalent modification
Allosteric regulation
Zymogens: Proenzymes or zymogens: Activated by
proteolysis

Km of enzymes in the range of

Proinsulin: Insulin
Chymotrypsinogen: Chymotrypsin
Blood clotting factors: Serine protease cascade leading to

fibrinogen to fibrin

Isozymes: Lactate dehydrogenase: A4, A3B1, A2B2, A1B3,

B4
Modular proteins: cAMP-dependent protein kinase: R2C2

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General properties of regulatory

proteins
Kinetic properties
Do not follow simple Michaelis-Menten kinetics
Activity sigmoidal: Higher order dependence on

substrate concentration
Cooperativity
Allosteric inhibition
Often regulated by activation
Oligomeric organization
Effectors alter distribution of conformational isomers
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Cooperativity models
Monod, Wyman, Changeux (1965): Symmetry model
Two conformations: T (tense or taut) and R (relaxed)
R state high affinity, T state lower affinity
Positive homotropic effectors
Substrate binding shifts equilibrium to R

Heterotropic effectors
Positive effector: Binds to R state and shifts equilibrium

toward R
Negative effector: Binds to T state and shifts equilibrium
toward T
Koshland, Nemethy, Filmer (1966) Sequential model:
Induced fit: S-binding induces conformational change
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