In the Classroom
Enzyme Catalysis and the Gibbs Energy
Addison Ault
Department of Chemistry, Cornell College, Mount Vernon, IA 52314; aault@cornellcollege.edu
I read with interest Brian Bozlee’s recent article “Refor-
mulation of the Michaelis–Menten Equation: How Enzyme-
Catalyzed Reactions Depend on Gibbs Energy” (1). In this
article he considers the paradoxical result of a change in the
energy of the enzyme–substrate complex, ES. “Is it possible
that raising G3 could both speed and slow the rate of product
formation?” (1, p 106).
We can distinguish two rates for an enzyme-catalyzed reac-
tion. One is the rate at low substrate concentration (extrapolated
to zero substrate concentration), and the second is the rate at
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high substrate concentration (extrapolated to infinite substrate
concentration). When we make this distinction we can resolve
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the paradox presented in Bozlee’s article.
Reaction of Interest
We can represent the reaction of interest (1) as
k1 k2
E+S ES E+P
k−1
where E is the enzyme, S is the substrate, and P is the product.
The unusual, but characteristic, feature of the typical enzyme-
catalyzed reaction is that the initial rate increases with increased
substrate concentration, but at high substrate concentrations
the initial rate approaches a maximum that is independent of
further increases in the concentration of the substrate. This
behavior is illustrated by the example in Figure 2 of Bozlee’s
article (1, p 107).
Gibbs-Energy Profile
The connections between rates and Gibbs energies are
often summarized as a plot of Gibbs energy versus “progress of
reaction”. While such a graph, or “Gibbs-energy profile”, is a vast
simplification of what actually happens, it does represent in a
useful way the average behavior of the reacting species. Bozlee
shows such a graph in Figure 1 of his article (1, p 106).
Kinetics of a Simple Enzyme-Catalyzed Reaction
The kinetic behavior of the reaction1 of interest can be
represented by the following equation in which [E0] is sum of
the concentrations of all forms of the enzyme, and [S] is the
concentration of unbound substrate (2–4):
1 to start at the “higher valley” because the great “pressure” of S
rate = k2 [E 0 ]
k−1 + k2 (1)
1 +
k1 [S ]
When the constants in the second term of the denominator are
combined and designated as KM,
k + k2
KM = −1 (2)
k1
the rate equation can be rewritten as
1 The experimental second-order rate constant, from eq 5,
rate = k2 [E 0 ]
K (3)
1 + M
[S ]
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Rate at High [S]
We can see that when [S] becomes high the term in the
parentheses, which is always fractional, approaches 1. Under
these conditions [E0] is equal to [ES] because [E] has been
driven down by the large excess of S, which converts E to ES.
The equation can then be rewritten as
rate = k2 [E 0 ] (4)
This corresponds to Bozlee’s eq 2, in which [E]T corresponds
to [E0] . However, where Bozlee says that “[E]T is the total con-
centration of bound and unbound enzyme” we must remember
that here [E]T is actually [ES] alone since, at high [S], “all” the
enzyme is present as ES and “none” is present as E.
This increase and then leveling-off of the initial rate with
increasing [S] is sometimes called a “saturation” effect; E be-
comes “saturated” with S.
Rate at Low [S]
When [S] is low the second term in the denominator of
eq 1 becomes large and eq 1 can then be rewritten as
k2 k1 [E 0 ][S ]
rate = (5)
k−1 + k2
Alternatively, eq 3 can be rewritten as
k2 [E 0 ][S ]
rate = (6)
KM
These equations indicate that when [S] is low the initial rate of
the enzyme-catalyzed reaction will be proportional to [S]. Under
these conditions, when [S] is low, [E0] is equal to [E] because
“all” the enzyme is present as E and “none” as ES.
Gibbs Energy at High [S]
In Figure 1 the vertical distance k2 is the Gibbs energy of
activation for the rate at high [S], conditions under which the
experimental first-order rate constant equals k2. The vertical dis-
tance k2 is the Gibbs energetic distance from the “higher valley”
to the “high pass” on the path to product. The reaction appears
when [S] is high forces S onto E to give ES; all E is ES.
Gibbs Energy at Low [S]
In Figure 1 the vertical distance k2k1/k‒1 is the Gibbs energy
of activation for the rate at low [S], conditions under which
the experimental second-order rate constant is approximately
k2k1/k‒1.
When Is This Approximation Valid?
is exactly k2k1/(k‒1 + k2), or, from eq 6, it is exactly k2/KM. The
approximation of the experimental second-order rate constant
as k2k1/k‒1 is valid when we can assume that the steady-state con-
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 In the Classroom
centration of ES is the same as what would be the equilibrium
concentration of ES if no ES could go on over the “high pass”.
Remembering that the Gibbs energy scale is a log scale, the
apparent 5-fold difference in vertical distance from the “higher
valley” to the transition state, ts2, (and on to product) and the
vertical distance from the “higher valley” to ts1 (and back to
starting materials) corresponds to a 100,000-fold difference in
rate constant. If we take k‒1 as relative 1, k2 would be relative
0.00001. Setting k2k1∙(k‒1  +  k2) equal to k2k1/k‒1 is then an
excellent approximation.
Why Do We Need a “Higher Valley”?
If there were no “higher valley”, and only the “high pass”,
the rate of the reaction would be proportional to [S] at all con-
centrations of S. We would not see the “saturation” effect that
characterizes enzymatic reactions.
How Important Is the Gibbs Energy
of the “Higher Valley”?
The “higher valley”, indicated by G3 in Bozlee’s Figure 1
(1,  p 106), simply divides the total Gibbs activation energy
under conditions of low [S] into two parts. The first part is the
Gibbs energy difference (k1/k‒1 in Figure 1) that corresponds to
the pre-equilibrium, actually steady-state, in which ES is formed.
The second part is the Gibbs activation energy (k2 in Figure 1)
that corresponds to the further reaction of ES, the product of the
pre-equilibrium. It really does not matter where the division is
made. The sum of the two parts will be the same; k2k1/k‒1 would
be unchanged because a change in k2 would be exactly balanced
by a compensating change in k1/k‒1.
How Important Is the Gibbs Energy
of the Transition State?
The Gibbs energy of ts1, indicated as G2 in Bozlee’s Figure 1
(1, p 106), does not enter the calculation. The Gibbs energy of
ts1 should be higher than the Gibbs energy of the “higher val-
ley” (otherwise there would be no valley), but not so high that
it approaches the energy of ts2. If the Gibbs energy of ts1 were
to approach the Gibbs energy of ts2 the rate of equilibration of
ts2
G4
k2
ts1
k2 k1 G2
Gibbs Energy
k∙1
G3
k1
k∙1
G1
k1 k2
E∙S ES
k∙1
Progress
Figure 1. Plot of Gibbs energy for the progress of a standard
reaction.
1070 Journal of Chemical Education  •  Vol. 86  No. 9  September 2009  •  www.JCE.DivCHED.org  •  © Division of Chemical Education
E with ES would decrease, and if the Gibbs energy of ts1 were to
exceed the Gibbs energy of ts2 the reaction of E with S to give ES
would become the rate-limiting step of the reaction.
Conclusions
These conclusions apply only to the very first part of the
reaction; they apply only to the initial rate, the first moments of
the reaction. The initial rate is typically calculated by extrapola-
tion back to zero time.
1. The rate-limiting process under all conditions is passage
through the transition state of highest Gibbs energy, the “high
pass”.
2. At high [S] the Gibbs energy of activation is the Gibbs energy
difference between the “higher valley” and the “high pass”. At
high [S] all E starts as ES from the “higher valley” to go to the
“high pass”. The “higher valley” acts as a “base camp” that gives
E a “head start” toward the “high pass”. All E gets pushed up
into the high valley as ES by the huge excess of S.
3. At low [S] the Gibbs energy of activation is the Gibbs energy
difference between the “lower valley” and the “high pass”. This
Gibbs energy difference equals the sum of the both the Gibbs
energy difference between the “lower valley” and the “higher
valley” and the Gibbs energy difference between the “higher
valley” and the “high pass”. At low [S] all E starts from the
“lower valley” to go to the “high pass.” At low [S] the concentra-
tion of ES at the “base camp” is negligible.
4. When [S] = KM the initial rate, as we can see from eq 3, will be
half of the maximum possible initial rate, the rate at high [S].
We account for this by saying that when [S] = KM only half of
the enzyme is present as ES. The implication is that the half of
the enzyme present as E makes a negligible contribution to the
rate under these conditions.
Summary
We can summarize in this way:
• One, above, is always true: the highest energy point on the path
to product is the “high pass”.
• Two, above, is the special case when [S] is so high that in the
steady state “all” the enzyme is present as [ES]. Here, the trip
over the highest pass starts from the “high valley”.
• Three, above, is the special case when [S] is so low that in the
steady state “all” the enzyme is present as [E]. This time the trip
over the highest pass starts from the “low valley”.
We see, then, that there are two “special” rates for an enzyme-
catalyzed reaction, a low (small [S]) rate and a high (large [S])
rate. We need to think of them separately.
Resolution of the Paradox
Realizing now that there are two extreme rates for an
enzyme-catalyzed reaction, a low (small [S]) rate, and a high
(large [S]) rate, we are ready to answer Bozlee’s question: “Is it
possible that raising G3 could both speed and slow the rate of
product formation?” (1, p 106).
E∙P
Effect on Vmax
As Bozlee says (1), the fast (high [S]) rate will increase Vmax
because this change will move the “higher valley”, or G3, closer
to the “high pass”, or G4. The difference between G3 and G4 will
be less; k2 will be greater.
 

Effect on the Initial Rate
There will, however, be no effect on the rate of the slow
(low [S]) rate because a change in G3 does not change the ener-
getic distance between the “low valley” and the “high pass”, the
difference between G1 and G4. The second-order rate constant
will still be k2k1/k‒1, which equals k2/KM, as indicated in eq 4.
Effect on KM
A slight upward adjustment of the “high valley”, a slight
increase in G3, will not only increase k2, but it will also increase
k‒1. Since k1 will not be changed by this adjustment, this adjust-
ment will also increase KM since
k
KM ≈ −1 (7)
k1
We can see this also by looking at the Gibbs-energy profile. If
the “high valley” is given a slight upward adjustment the ES/E
ratio in the steady state will become less favorable, and a greater
concentration of substrate will be required to bring the rate up
to half of the new, higher Vmax. Bozlee’s Figure 2 illustrates this
nicely (1, p 107).
You Cannot Get Something for Nothing
Although a slight upward adjustment of the “high valley”
will increase Vmax, which can be perceived to be a “benefit”, there
will be, however, an associated “cost”. That cost is the need for a
greater concentration of substrate S to raise the rate of the reac-
tion to any particular fraction of Vmax. Thus to achieve a certain
rate, determined by the height of the highest pass, there can be
different combinations of concentration and rate constant, and
the price of a more favorable rate constant is the requirement
for a higher concentration.
Comparison with Acid Catalysis
We see this effect in many reactions, and the acid-catalyzed
hydrolysis of an ester is a familiar example. As the reaction
mixture is made more acidic, the reaction speeds up because in
a more acidic solution there is a higher concentration of a more
reactive species, the “ester–proton complex”. The price paid for
this benefit is a higher concentration of hydronium ion, a price
the chemist is quite willing to pay. However, since esters are only
weakly basic, we do not observe “saturation”.
Pedagogy
Three basic ideas must be kept in mind while thinking
about connections between the rates of reactions and the Gibbs
energies of intermediates and transition states.
• The rate and the rate constant are not the same thing.
The rate is one or more concentrations times the rate con-
stant.
That is,
rate = concentration rate
constant
or
rate = how how
much well
© Division of Chemical Education  •  www.JCE.DivCHED.org  •  Vol. 86  No. 9  September 2009  •  Journal of Chemical Education
In the Classroom
Jencks (5) puts it this way:
As in many kinetic descriptions, it is important to make a clear
distinction between a rate constant and the absolute rate for
a particular step; the latter quantity is a function of the rate
constant for that step, the rate and equilibrium constants for
preceding steps, and the concentrations of the reactants. This
point may seem obvious, but uncertainty regarding the mean-
ing of “rate-determining step” has repeatedly led to confusion
in description of reaction mechanism.
• The rate-determining step is passage over the highest pass on
the path to product.
We quote again from Jencks (5):
it is the highest point on the overall Gibbs-energy profile, not
the highest energy barrier for an individual step, that deter-
mines which step is rate-determining.
• In the next sentence Jencks (5) tells us why it is k2/k‒1 and not
k1/k‒1.
Thus, under steady-state conditions in which the intermedi-
ate does not accumulate, the critical factor in determining
which step of a reaction is rate-determining is the relative rate
of breakdown of the unstable intermediate in the forward
and reverse direction, that is, the ratio k2/k‒1 rather than the
relative rates of formation and breakdown of the intermediate,
k1/k‒1.
Note
1. In this simple model we assume the absence of any inhibitor,
and so we do not include [I], [EI], or [EIS] in our calculations (2–4).
When we consider the forms of the enzyme, E, we can then say that the
enzyme will be present only as the free enzyme, E, and as the enzyme
substrate complex, ES. When this is true the total concentration of all
forms of the enzyme, [E0], equals [E] + [ES].
We now consider the forms of the substrate, S. The substrate can
be present as free substrate, S, bound substrate, ES, and, eventually,
as product, P. Thus, in general and in the absence of an inhibitor, the
concentration of all forms of the substrate, [S0], equals [S] + [ES] + [P].
Even though some substrate will be present as ES the concentration of
the substrate, [S], is typically orders of magnitude greater than the con-
centration of the enzyme. Therefore, even under saturating conditions,
[ES] will be orders of magnitude less than [S]. It is for this reason that
the concentration of free substrate, [S] is typically taken to be equal to
the total concentration of substrate, [S0] (2).
Restricting ourselves, however, to “initial rates” we can assume
that [P] = 0.
Literature Cited
Bozlee, B. J. J. Chem. Educ. 2007, 84, 106–107.
1.
Shaw, W. H. R. J. Chem. Educ. 1957, 34, 22–25.
2.
Ault, A. J. Chem. Educ. 1974, 51, 381–386.
3.
Ault, A. J. Chem. Educ. 2008, 85, 1432–1434; online supplement
4.
pp 15–16.
5. Jencks, W. P. Catalysis in Chemistry and Enzymology; McGraw-
Hill Book Company: New York, 1969; p 475.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Sep/abs1069.html
Abstract and keywords
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