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Lecture 2

Enzyme Kinetics
Introduction
Enzymes are Biological Catalysis
A catalyst is a substance that increases the rate (velocity) of a
chemical reaction.
Most biological catalysts are proteins.
The material acted upon by the catalyst is the substrate.
Although a catalyst participates in the reaction process, it is
unchanged after the process is complete.
A catalyst increases the rate at which a reaction reaches
equilibrium but does not alter Keq or Go' for the reaction.
A thermodynamically favorable process is not made more
favorable by the presence of a catalyst.
A thermodynamically unfavorable process is not made
favorable by the presence of a catalyst.
Kinetics

The Rate Constant


For the irreversible reaction A B.
This is a first order reaction (there is only a single
reactant).
The velocity (v) or reaction rate is given by the
rate of formation of product or the rate of
disappearance of reactant.
The velocity (v) or reaction rate is, where k= the
rate constant.
For the reaction A + B products, v = k[A][B].
This is a second order reaction (there are two
reactants).
Reaction Rate Theory

What determines the rate of a


reaction?
For every reaction there is a high
energy transition state through
which the reactants must pass in
order for the reaction to occur.
The height of the energy barrier,
Go, determines the rate of
the reaction.
k=Qe(-Go/RT)
Q is a collection of constants.
Catalysis
A catalyst functions by lowering the
activation energy for a reaction by
amount = Go.
A catalyst does not alter the G for
the reaction.
Go = Ho -TSo - a catalyst can
accelerate a reaction by affecting
either Ho or So, or both.
Strong binding of the transition state
to the catalyst lowers Ho - makes it
more negative.
Proximity and orientation of the
substrates on the catalyst favor
formation of the transition state by
reducing So.
Enzymes
Enzymes are highly effective catalysts that carry
out complex chemical transformations under mild
conditions (water, neutral pH).
Enzymes show great specificity with regard to the
reactions they catalyze and the substrates they
react with.
Enzymes can be regulated.
Enzymes carry out their catalytic role by binding
the substrate to a specific area of the protein
called the active site. (Companion:
Enzymes/Enzyme Kinetics).
Several amino acid side chains comprise the
active site.
Coenzymes

Coenzymes are
small organic
molecules, derived
from vitamins that
participate in the
chemical reactions
catalyzed by many
enzymes.
Coenzymes
Coenzyme Vitamin Reaction Mediated

Biotin Biotin Carboxylation


Cobalamin B12 Alkylation
Coenzyme A Pantothenic acid Acyl Transfer
Flavin Riboflavin Oxidation-Reduction
Lipoic Acid Lipoamide Acyl Transfer
Nicotinamide Niacin Oxidation-Reduction
Pyridoxal Phosphate Pyridoxal Amino Group Transfer
Tetrahydrofolate Folate One-Carbon Group Transfer
Thiamine Pyrophosphate Thiamine Aldehyde Transfer
Summary of factors responsible for the rate enhancement
seen with enzyme catalysis
Uncatalyzed reactions in solution can be slow because
They involve the formation of unstable positive and negative
charges in the transition state.
They frequently require several molecules to be brought
together with a concomitant loss of entropy.

These difficulties are lessened with enzymes because


Strategically placed acids, bases, metal ions, or dipoles that
are part of the structure of the enzyme stabilize charges.
Covalent catalysis is used to give reaction pathways of lower
energy.
Entropy losses are minimized because the necessary catalytic
groups are part of the enzyme structure.
These features are paid for in two ways.
The original synthesis of the enzyme costs energy,
although the enzyme is used repeatedly.
The enzyme-substrate binding energy is used to
immobilize the substrate at the active site and hold it
next to the catalytic groups.

This binding energy is inherently available for use but


it is generally not utilized in uncatalyzed reactions.
Enzyme Kinetics

The simplest enzyme mechanism involves the


following two steps:

The rate of the enzymatic reaction is:


v = k1[ES]
In order to derive a
useful equation
describing this reaction
we make the steady-
state assumption,
which assumes that
over most of the
reaction course [ES] is
small and does not
change, i.e., d[ES]/dt=0.

Using this assumption, one can derive the


Michaelis-Menten equation.
Plotting Kinetic Data
The Michaelis-Menten
equation describes a
rectangular hyperbola.
The enzyme is
characterized by two
constants: KM and Vmax
Vmax is the maximal
rate of the reaction
which occurs when [S]
>> KM
KM is the substrate
concentration that
gives 1/2 maximal
velocity.
For determination
of KM and Vmax a
linear
transformation,
the Lineweaver-
Burk plot, is
useful.
Turnover Number
The turnover number of an enzyme, kcat, is the maximal
velocity per enzyme molecule per unit of time.

Enzyme Efficiency
We can rewrite the rate equation as:

When [S] << KM, then kcat/KM is a second order rate


constant, and is a measure of the efficiency of the enzyme
at low [S].
The maximal value of kcat/KM is 108-109, which is diffusion-
controlled.
Enzyme Substrate kcat (sec-1) KM(M) kcat/ KM (M-1) (sec-1)

Catalase H2O2 4.0x107 1.1 4.0x107

Carbonic anhydrase CO2 1.0x104 1.2x10-2 8.3x107

Acetylcholine esterase Acetylcholine 1.4x104 9.0x10-5 1.6x108

Fumarase Fumarate 8.0x102 5.0x10-6 1.6x108

For different substrates, kcat/KM is also the best way to determine the
specificity of an enzyme.
For hydrolysis of a peptide bond by the
proteolytic enzyme chymotrypsin, the nature
of the R1 side chain is critical.

R1 kcat/KM (M-1sec-1)
Gly 1.3x10-1
Val 3.6x102
Leu 3.0x103
Phe 1.0x105

The Phe-containing substrate is best!


Enzyme Regulation

Amount of enzyme (transcriptional).


Amount of substrate.
Control of activity.
Allosteric regulation.
Covalent modification.
Inhibitors.
Negative-feedback pathway
Allosteric Regulation (remember hemoglobin!!).
Multisubunit enzymes

Homoallostery -
cooperative
substrate binding
and
activation.
Heteroallostery
regulation by effector
molecules, which can be
positive or negative.
Allosteric effectors bind
at a site different from
the active site.Allosteric
effectors can activate
(favor R state) or inhibit
(favor T state).
Reversible
covalent
modification is
widely used to
regulate enzyme
activity.
Phosphorylation
is a common
reaction.
Irreversible
covalent
modification.
Many enzymes
are made as
inactive precursors,
zymogens.
Activation of the
zymogen involves
proteolytic
cleavage and in this
case (trypsinogen)
removal of a
peptide fromthe
amino terminus.
Enzyme Inhibitors
The use of enzyme inhibitors can often provide valuable
information about an enzymatic mechanism. Many drugs
are based on the use of enzyme inhibitors, e.g., penicillin
inhibits an enzyme involved in bacterial cell wall synthesis.
A competitive inhibitor competes with the substrate for
binding at the active site, increasing Km.
Competitive inhibitor
A noncompetitive inhibitor binds to a site other
than the active site and inhibits product
formation. Noncompetitive inhibitors decrease
velocity, including Vmax, by decreasing kcat.
Allosteric inhibitor
Covalent Inhibition
Irreversible or covalent
inhibition involves chemical
modification of the protein.

Compound I bound to serotype 2 human rhinovirus 3C protease. The protein is rendered as


a semitransparent solvent-accessible surface with associated protein backbone and side-
chain atoms colored pink. Catalytic triad residues are blue. Red spheres represent ordered
solvent molecules. Inhibitor atoms are colored green for carbon, blue for nitrogen, and red
for oxygen. The inhibitor carbon covalently bonded to Cys-147 is highlighted in light green.
Effect of pH

pH is not an important
regulatory mechanism,
but the effect of pH can
be highly informative
about the mechanism.
Changing pH can increase
or decrease the rate.
This pH-rate profile
suggests that a
deprotonated histidine is
involved in the catalytic
step.
This pH-rate
profile suggests
that a
deprotonated
histidine is
involved in the
catalytic step.
This pH-rate
profile suggests
that a
protonated
lysine is
involved in the
catalytic step.
Note that the apparent pKa derived from inspection of kinetic data
may be significantly different than the actual pKa of the side chain.
More sophisticated analysis is required to obtain an accurate
estimation of the pKa in the enzyme.