Lectures - Biochemistry 1 - 2021-2022 Prof Version 6.01 - 23-24-231-246

Transition States and Reaction Rates
We now consider two strategies for increasing reaction rates:

(1) raising the temperature of the reaction, and
(2) lowering the free energy of the transition state. The standard free energy of activation, ΔG∙‡, represents the
additional free energy (above the average free energy of reactant molecules) that molecules must have to attain the
transition state.
If the activation barrier to the reaction is high, only a small fraction of the molecules will have enough energy to
surmount it, or only a small fraction of collisions will be energetic enough for the reaction to occur.
Transition State Theory Applied to Enzymatic Catalysis
We now consider two strategies for increasing reaction rates:

(1) raising the temperature of the reaction, and
(2) lowering the free energy of the transition state. The standard free energy of activation, ΔG∙‡, represents the
additional free energy (above the average free energy of reactant molecules) that molecules must have to attain the
transition state.
If the activation barrier to the reaction is high, only a small fraction of the molecules will have enough energy to
surmount it, or only a small fraction of collisions will be energetic enough for the reaction to occur.
transition state theory assumes that a reactant molecule that attains the transition state rapidly decomposes to a lower-
energy state, such as the product state, or to an intermediate state (see below). Because bonds are in the process of
breaking and/or forming in the transition state, the lifetime of the transition state is similar to the vibrational
frequencies of covalent bonds—on the order of a picosecond (10-12 s). These concepts are summarized in the following
general expression for the rate constant k:
where T is temperature, R is the gas constant, and A is a factor that accounts for the vibrational frequency of bonds in
the transition state. At 310 K (37 °C), A has a value of ≈ 6.4 * 1012 s-1.
The rate enhancement is the ratio of the rate constants for the catalyzed (kcat) and the noncatalyzed (knon) reactions
for a given set of conditions (e.g., temperature, pH). The rate enhancement indicates how much faster the reaction
occurs in the presence of the enzyme.
For example, we can compare the rate of amide bond hydrolysis by the enzyme carboxypeptidase A versus the
noncatalyzed rate at pH = 8 and 23 °C:
The rate enhancement for an enzyme-catalyzed reaction is

the ratio of the rate constants for the catalyzed (kcat) and the
noncatalyzed (knon) reactions. The rate enhancement
indicates how much faster the reaction occurs in the
presence of the enzyme.
We can combine previous equations evaluate by how much an enzyme must stabilize the transition state to
achieve these observed rate enhancements:
FIGURE Effect of a catalyst on activation energy. A free energy diagram (blue curve) is shown, along with an
alternative catalyzed path (red curve) for the reaction. The catalyst lowers the standard free energy of activation, ΔG°‡,
and thereby accelerates the rate because more of the reactant molecules have the energy needed to reach this
lowered transition state. The rate enhancement is related to ΔΔG°‡. Note that the values of ΔG°A→B for both the catalyzed
and noncatalyzed reactions are the same; thus, the reaction equilibrium is not perturbed by the presence of the
catalyst.
How Enzymes Act as Catalysts: Principles and Examples
the role of a catalyst is to decrease ΔG°‡ by facilitating the formation of the transition state. In this section, we describe
some common strategies used by enzymes to achieve a reduction in ΔG°‡, and then illustrate those strategies with two
examples: lysozyme and chymotrypsin.
FIGURE Importance of intermediate states. An enzyme

may alter the reaction pathway to one that includes one
or more intermediate states that resemble the transition
state but have a lower free energy (red curve).
In the case of a single intermediate, the activation
energies for formation of the intermediate state and for
conversion of the intermediate to product (ΔG°‡1 and ΔG°‡2,
respectively) are lower than the activation energy for the
uncatalyzed reaction (blue curve). In this figure, only
activation energies for the enzyme-catalyzed forward
reaction (A → B) are shown.
How Enzymes Act as Catalysts: Principles and Examples
THE KINETICS OF ENZYMATIC CATALYSIS
Reaction Rate for a Simple Enzyme-Catalyzed Reaction: Michaelis–Menten Kinetics
the reaction rate, or velocity, defined as the observed rate of formation of products, can be
expressed as:
kcat is an aggregate rate constant = k2k3/(k2 + k3). In the limiting case where k3 >> k2, kcat ≈ k2.
In the steady state, the rates of formation and breakdown of ES are equal. Therefore,
Combining the ratio of rate constants in
Equation above gives a single constant,
KM:
At this point, [ES] is expressed in terms of [E] and [S]. To get [E]t into the equation, rather than [E], recall from
Equation 8.20 that [E] = [E]t - [ES]. Putting this into previous equation yields:
This rearranges to
Finally, inserting this result into Equation 8.19 gives an expression for ν in terms of [E]t and [S]:
This equation is called the Michaelis–Menten equation, and

KM the Michaelis constant.
FIGURE Reaction velocity as a function of substrate concentration.

This graph, a plot of Equation 8.30, shows the variation of reaction velocity with substrate concentration according to
the Michaelis–Menten model of enzyme kinetics. Here, [S] is given in terms of K M. At the point where [S] = KM, the
reaction has exactly half its maximum velocity.
The values of v plotted here are determined from the initial rates of the reaction
Previous equation will reach its maximum value. When [S] >> KM, KM + [S] ≈ [S], and rate Equation simplifies to the
expression for Vmax:
Thus, kcat[E]t in Equation 8.28 is equivalent to Vmax, and the Michaelis–Menten rate equation is:
This is the most familiar form of the Michaelis–Menten

equation.
FIGURE Reaction velocity as a function of substrate concentration.

This graph, a plot of Equation 8.30, shows the variation of reaction velocity with substrate concentration according to
the Michaelis–Menten model of enzyme kinetics. Here, [S] is given in terms of K M. At the point where [S] = KM, the
reaction has exactly half its maximum velocity.
The values of v plotted here are determined from the initial rates of the reaction
Analysis of Kinetic Data: Testing the Michaelis–Menten Model
FIGURE Analysis of initial rates.

Analysis of Kinetic Data: Testing the Michaelis–Menten Model
Given such data for concentrations and initial rates, how are KM and kcat calculated? In practice, modern nonlinear
curve-fitting software can fit the data plotted in previous figure (b) directly to provide these parameters. Before such
data analysis became widely available, a different method was (and still is) used. Equation 8.30 can be rearranged to
give the expression for a linear graph. Several kinds of graphs are possible, but it is most common to use a double
reciprocal plot, also called a Lineweaver–Burk plot (FIGURE).
A Lineweaver–Burk plot provides a quick test for adherence to Michaelis–Menten kinetics and allows easy evaluation
of the critical constants. It also allows discrimination between different kinds of enzyme inhibition and regulation.
Taking the reciprocal of both sides of Equation 8.30, we find:
FIGURE A Lineweaver–Burk plot. In this double reciprocal plot, 1/v is

graphed versus 1/[S], according to the previous equation. Note that a
linear extrapolation of the data gives both V max and KM.
Enzyme Inhibition
A major distinction must be made between reversible inhibitors and irreversible inhibitors.
Reversible Inhibition
The various modes of reversible inhibition all involve the noncovalent binding of an inhibitor to the enzyme, but they
differ in the mechanisms by which they decrease the enzyme’s activity and in how they affect the kinetics of the
reaction.
Competitive Inhibition
Consider a molecule that can bind to an enzyme active site because it is structurally similar to the true substrate for the
enzyme. If this molecule can also be processed by the enzyme, it is merely a competing alternative substrate. However,
if the molecule binds to the active site but cannot undergo the chemical conversion step, it effectively reduces the
enzyme’s availability to carry out chemistry on true substrates. Such a molecule is called a competitive inhibitor because
it competes with the substrate for binding to the same site on the enzyme (FIGURE).
Enzyme Inhibition
Enzyme Inhibition
Enzyme Inhibition

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Transition States and Reaction Rates

We now consider two strategies for increasing reaction rates:

We now consider two strategies for increasing reaction rates:

The rate enhancement for an enzyme-catalyzed reaction is

FIGURE Importance of intermediate states. An enzyme

This equation is called the Michaelis–Menten equation, and

FIGURE Reaction velocity as a function of substrate concentration.

This is the most familiar form of the Michaelis–Menten

FIGURE Reaction velocity as a function of substrate concentration.

FIGURE Analysis of initial rates.

FIGURE A Lineweaver–Burk plot. In this double reciprocal plot, 1/v is

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