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Biochemistry 462a - Enzyme Kinetics

Reading - Chapter 8
Practice problems - Chapter 8: (not yet assigned); Enzymes extra problems


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
• 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
• A thermodynamically unfavorable process is not made favorable by the presence of a


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
The velocity (v) or reaction rate is, where k= the rate

For the reaction A + B → products, v = k[A][B]. This is a second order reaction (there are two

Reaction Rate Theory

• What determines the rate of a

• 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.


• 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‡ -T∆So‡ - a catalyst
can accelerate a reaction by
affecting either ∆Ho‡ or ∆So‡, or
• 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 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 are small organic molecules, derived from vitamins that participate in the chemical
reactions catalyzed by many enzymes.

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

o They involve the formation of unstable positive and negative charges in the transition
o They frequently require several molecules to be brought together with a concomitant
loss of entropy.
• These difficulties are lessened with enzymes because
o Strategically placed acids, bases, metal ions, or dipoles that are part of the structure of
the enzyme stabilize charges.
o Covalent catalysis is used to give reaction pathways of lower energy.
o Entropy losses are minimized because the necessary catalytic groups are part of the
enzyme structure.
• These features are paid for in two ways.
o The original synthesis of the enzyme costs energy, although the enzyme is used
o 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:

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
o Vmax is the maximal rate of the
reaction which occurs when [S] >>
o 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

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 CO2 1.0x104 1.2x10-2 8.3x107


Acetylcholine 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 sidechain 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.
o Allosteric regulation.
o Covalent modification.
o Inhibitors.

Allosteric Regulation (remember hemoglobin!!).

• Multisubunit enzymes

Homoallostery - cooperative substrate binding and


• 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 of a Ser is a common

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.

• 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.

Covalent Inhibition

Irreversible or covalent inhibition involves

chemical modification of the protein.

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 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 sidechain. More sophisticated analysis is required to obtain
an accurate estimation of the pKa in the enzyme.