Research: Science and Education

Rapid-Equilibrium Enzyme Kinetics

Robert A. Alberty
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; alberty@mit.edu

Rapid-equilibrium rate equations for enzyme-catalyzed
reactions are especially useful because if experimental data can
be fit by these simpler rate equations, the Michaelis constants can
be interpreted as equilibrium constants. However, for some reac-
tions it is necessary to use the more complicated steady-state rate
equations. Rapid-equilibrium derivations of rate equations have
been described by Cha (1) and Segel (2). These equations can be
derived for ordered mechanisms by the algebraic method, but
there is a problem in deriving rate equations for random mecha-
nisms, that is, mechanisms with more than one pathway. This
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fifteen complete rapid-equilibrium rate equations. More rate
equations can be constructed from these half-reactions, and the
number of half-reactions can be increased.
Relation Between Number of Reactants,
Number of Components, and Number of Reactions
in Equilibrium Calculations
In calculating the equilibrium composition for a biochemi-
cal reaction system at a specified pH, apparent equilibrium con-

problem is often solved by using the King–Altman (3) method stants, K', are used. The number of reactants (sums of species)
to derive the complete steady-state rate equation for the random in the system, N', the number of independent reactions R', and
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mechanism and then eliminating certain terms, as described by the number of components, C', has to satisfy (6) the following
Cleland (4) and Roberts (5). The problem in applying the alge- relationship:
braic method to mechanisms with more than one pathway arises
because there is a fundamental difference between kinetics and
thermodynamics. Thermodynamics is involved because in the
rapid-equilibrium method the assumption is made that all the
reactants prior to the rate-determining step are at equilibrium.
Random mechanisms have alternate pathways such as
B B
N b  R b  C b (5)
Components are the things that are conserved. In chemical ther-
modynamics, atoms of elements are conserved, but, even there,
if atoms of two elements are always in the same ratio whenever
they occur in reacting species, the atoms of the two elements
count as one element. Specifying the pH creates pseudoisomer
  groups (for example, ATP4‒, HATP3‒, and H2ATP2‒) that count
E A EA as a single reactant. The issue of numbers of components comes
(1)
up when cycles are postulated, as in eq 1. Alternate pathways
are not permitted in thermodynamics (or in rapid-equilibrium
kinetics) because the four equilibrium constants in eq 1 are not
EB  A EAB products
independent. Therefore, in calculating the equilibrium compo-
sition, one of these four reactions has to be omitted so that N'
where A and B are reactants; E is the enzyme; and EA, EB, and
= 6, C' = 3 (E, A, B), and R' = 3. The choice of components is
EAB are transient complexes. The double arrows, , represent
optional, but the number of components C' is not.
reversible reactions that may be fast or slow, but the single arrow
Equation 5 can be understood in the following way. The
represent an irreversible reaction. Thermodynamics requires that
basic objective of the thermodynamics of reaction systems is to
the reactions used in an equilibrium calculation must be inde-
calculate the equilibrium composition. For biochemical reaction
pendent. The four reactions in the alternate pathway in eq 1 are
systems, the pH has to be specified; and perhaps pMg needs to
not independent because the equilibrium constant for any one of
be specified. There are equilibrium equations for R' reactions
them can be calculated from the other three. The following three
and a conservation equation for each of C' components. This
reactions can be used to calculate the equilibrium composition
provides just enough information to calculate the equilibrium
in the rapid-equilibrium treatment of the mechanism in eq 1:
concentrations of N' reactants. The number of reactants, number
EA EA of components, and number of independent reactions prior to
(2)
the rate-determining reaction for five mechanisms of enzyme-
catalyzed reactions are given in Table 1. These mechanisms are
EB EB (3) referred to as half-reactions because a forward half-reaction
can be combined with a reverse half-reaction to obtain the
EA  B EAB products (4)
The double arrows, , are used in the reactions that are as-
sumed to be equilibrated rapidly. Note that EB + A EAB is
not included. Since the issue of the independence of reactions
in equilibrium calculations is so important, the next section
provides more information.
This article describes a new method for obtaining rapid-
equilibrium rate equations by combining half-reactions. Any
forward half-reaction can be combined with any reverse half-
reaction. Five half-reactions are described and used to produce
1136 Journal of Chemical Education  •  Vol. 85  No. 8  August 2008  •  www.JCE.DivCHED.org  •  © Division of Chemical Education
mechanism of a complete reaction. Notice that in each of these
mechanisms for rapid-equilibrium calculations, there is a single
reaction for each enzyme–substrate complex that is formed.
The half-reactions in Table 1 are similar to the half-reac-
tions of electrochemistry in that any two can be combined. In
electrochemistry the two half-reactions have to involve the same
number of electrons, and in rapid-equilibrium kinetics the two
half-reactions have to have the same denominator terms.
The shortest mechanism for the reaction A + B + C →
products involves only three reactions and is referred to here
as the ordered mechanism. A mechanism may be neither
 
 Research: Science and Education

completely ordered nor completely random. The most ran- Table 1. Number of Reactants N', Number of Components C’,
dom mechanism involves seven reactions. Between these two and Number of Reactions R’ for Rapid-Equilibrium Treatments
of Half-reactions
extremes, reactions involving EB, EC, EAC, and EBC can be
added. When one of these reactants is added, the number of Half-Reactions N’ C’ R’
reactants is increased by one and the number of reactions is
Simple mechanism 3 2 (E, S) 1
increased by one; therefore, the number of components is not E+S ES
changed. As a simplification, only the five mechanisms of half-
reactions in Table 1 are discussed. Ordered mechanism 5 3 (E, A, B) 2
In the next three sections the rapid-equilibrium rate equa- E+A EA
tions are derived for (i) ordered A + B P, (ii) random A + B EA + B EAB
P, and (iii) ordered A + B ordered P + Q, where P and Q
are products. Then a shorter way to derive rapid-equilibrium rate Random mechanism 6 3 (E, A, B) 3
equations based on the half-reactions in Table 1 is described and E+A EA
is applied to derive rapid-equilibrium rate equations for fifteen E+B EB
mechanisms of enzyme-catalyzed reactions. EA + B EAB

Ordered mechanism 7 4 (E, A, B, C) 3

Rapid-Equilibrium Rate Equation E+A EA
for Ordered A + B P EA + B EAB
EAB + C EABC
The ordered mechanism for A + B P involves four
steps: Random mechanism 11 4 (E, A, B, C) 7
<E >< A > E+A EA
EA
EA KIA  (6) E+B EB
< EA> E+C EC
EA + C EAC
<EA><B> <E >< A ><B > EB + C EBC
EA  B
EAB KB   (7)
<EAB> KIA < EAB> EA + B EAB
EAB + C EABC

EAB EP (8)

equation with eq 10 yields the following rapid-equilibrium rate

EP EP KP 
<E ><P> (9)
equation,
<EP > Vf < A ><B > V <P >
 r
where indicates that the reaction is equilibrated rapidly, and K IA KB KP
v  (12)
indicates the rate-determining reaction. The nomenclature for
1 
< A>

< A>< B >

Michaelis constants follows Cleland (4). When thermodynam-
ics is used to derive the equilibrium concentrations of E, EA,
EAB, and EP, the reactants EAB and EP count as one reactant
because they are pseudo-isomers. This was mentioned after eq 5
as applying both to chemical thermodynamics and biochemical
thermodynamics at specified pH. The total concentration of
enzymatic sites is given by
<P >
KIA KIA KB KP
where Vf = kf[E]t and Vr = kr[E]t. The four terms in the denomi-
nator correspond with E, EA, EAB, and EP. The steady-state
rate equation for the mechanism in eqs 6–9 has six terms in
the denominator (4). If kinetic experiments show that there
are only four terms in the denominator, the rapid-equilibrium
rate equation in eq 12 represents the experimental data and the
< E >t  <E >  <EA >  <EAB>  < EP > Michaelis constants are equilibrium constants. The Haldane
equation is given by
 <E> 1 
< A> 
< A><B> 
<P > (10)
KIA KIA KB KP V f KP <P >eq
K b A  B P
  (13)
Vr K IA KB < A>eq <B>eq
The rapid-equilibrium reaction rate v is given by
Reactions of the type A + B P are very common in
v  kf <EAB>  kr <EP > biochemical kinetics because H2O does not count as a reactant
in N'. The concentration of H2O does not change during a
kf < A ><B ><E > kr <P >< E > (11)
reaction, and [H2O] is not used in the rate equation. However,
 
KIA KB KP the standard transformed Gibbs energy of formation of water
Δf G′°(H2O) is involved in the calculation of the apparent equi-
where kf is the rate constant for the forward reaction and kr is librium constant K', and the hydrogen atoms of H2O do count
the rate constant for the reverse reaction. Replacing [E] in this in calculating the change in binding of hydrogen ions, Δr NH,

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 Research: Science and Education

in an enzyme-catalyzed reaction. In a list of 229 reactions for The total concentration of enzymatic sites is given by
which K' and Δr NH have been calculated at pHs 5, 6, 7, 8, and
9 at 298.25 K and 0.25 M ionic strength, half of the reactions < A> < A ><B>
involve H2O as a reactant (6). < E >t  <E > 1  
K IA KIA KB
(22)
Rapid-Equilibrium Rate Equation

<Q > 
<P > <Q >
for Random A + B P
K IQ K IQ KP
The random mechanism involves the four steps of the
preceding mechanism plus one more step: The rapid-equilibrium rate equation (5, 7) is

EB EB

KIB 
<EB > (14)
<E ><B> Vf < A><B > Vr <P ><Q >

KIA KB KP KIQ
v  (23)
The step EA + B EAB is not included because it is redundant
1 
< A >  < A ><B >  <Q >  <P> <Q >
in the calculation of equilibrium compositions. The total con- KIA KIA KB KIQ KIQ KP
centration of enzymatic sites is given by

[A] and [B] play different roles in the denominator, and so it is

< A> <B > < A ><B > <P> (15) possible to determine experimentally which reactant is bound
< E >t  <E > 1     first by the enzymatic site. A similar comment applies to the
K IA K IB K IA K B KP
reverse reaction. The Haldane relation is

kf KP KIQ <P >eq <Q >eq

Equation 11 applies and substituting eq 15 yields the following K b A  B PQ
  (24)
rapid-equilibrium rate equation: kr KIA KB < A >eq <B>eq
Vf < A>< B > V <P >
 r
K IA KB KP Rapid-Equilibrium Rate Equations for Half-reactions
v  (16)
1 
< >  < >  < ><B >  <P >
A B A
There is another way to derive rapid-equilibrium rate equa-
KIA K IB KIA KB KP tions for reactions and that is to combine rapid-equilibrium rate
equations for half-reactions. Any forward half-reaction can be
The effect of introducing randomness in the binding of A and B combined with any reverse half-reaction. For example, when the
is to add a term in [B] in the denominator. The effects of [A] and simple mechanism applies to the forward and reverse reactions
[B] are treated in the same way in eq 16, and this indicates they for S P, the rapid-equilibrium rate equation is
are bound randomly. Since the change is only in the denomina-
tor of eq 16, the Haldane equation in eq 13 is not changed. Vf < S > V <P >
 r
KS KP
v  (25)
Rapid-Equilibrium Rate Equation for the Mechanism
1 
< S >  <P >
for Ordered A + B Ordered P + Q KS KP
This ordered mechanism is given by
This rate equation can be written as
E  A EA
<E >< A >
KIA  (17)
< EA> V f <S > Vr <P >
KS KP
< EA><B>  <E >< A ><B> v  
EA  B EAB KB  (18) 1 
< >  < > 1  < >  <P >
S P S (26)
<EAB> K IA <EAB> KS KP KS KP
 v f  vr
EAB EPQ (19)
where vf is the rate of the forward half-reaction and vr is the rate
of the reverse half-reaction. The rate equation for the forward
EPQ EQ  P KP 
< EQ ><P > 
<E ><P><Q > (20) reaction must have a denominator term for the reverse reaction
<EAB> KIQ <EPQ > because the denominator terms describe the distribution of en-
zymatic sites between E, ES, and EP at equilibrium. Equation 26
EQ EQ KIQ 
< E > <Q > is the key to the use of half-reactions to derive the rate equation
(21)
<EQ > for a complete reaction.

1138 Journal of Chemical Education  •  Vol. 85  No. 8  August 2008  •  www.JCE.DivCHED.org  •  © Division of Chemical Education 
 Research: Science and Education

Table 2. Mechanisms of Enzyme-Catalyzed Reactions and Properties of Their Rapid-Equlibrium Rate Equations
N’ C’ R’ D Single Double Triple
Simple mechanism 5 2 3 3 2 0 0
E+S ES EP E+P
Ordered A + B P 7 3 4 4 2 1 0
E+A EA
EA + B EAB EP E+P
Random A + B P 8 3 5 5 3 1 0
E+A EA
E+B EB
EA + B EAB EP E+P
Ordered A + B Ordered P + Q 9 4 5 5 2 2 0
E+A EA
EA + B EAB EPQ EQ + P
EQ E+Q
Note: D is the number of terms in the denominator of the rate equation. Single, double, and triple refer to the numbers of terms
in the denominator involving 1, 2, and 3 reactant concentrations. The expanded version of Table 2 is available in the online
supplement.

The rapid-equilibrium rate equations for the five half- This last equation is given by Cornish-Bowden (7) as the initial
reactions in Table 1 are as follows: rate in the absence of products, but with different symbols for
Michaelis constants.
VS <S >
simple KS Rapid-Equilibrium Rate Equations
: v  (27) for Enzyme-Catalyzed Reactions Constructed
mechanism
1 
< S> with Rate Equations of Half-reactions
KS
As eq 26 shows, this in not quite as simple as taking the
Vf < A><B > difference between the rate equations for the two half-reactions.

Before the difference vf − vr is taken, the denominators of both
ordered K IA KB vf and vr must be augmented to account for all the enzyme–
: v  (28)
mechanism
1 
< A>  < A><B> substrate complexes at equilibrium. This has been demonstrated
K IA KIA KB for S P in the previous section.
The mechanisms considered in this article are given in
Table 2. This table gives N', C', and R', the numbers of terms
in the denominator of the rate equation, D, and the number
Vf < A ><B > of denominator terms with single concentrations, a product of
random KIA KB two concentrations, and a product of three concentrations in the
: v  (29) denominator. The numbers N', C', and R' for each rapid-equilib-
mechanism
1 
< A >  <B>  < A ><B > rium mechanism show that the mechanism has the correct num-
KIA KIB KIA KB ber of reactions in the mechanism. The number of components
C' is always equal to the number of reactants (A, B, C, P, Q, R)
Vf < A ><B ><C> in the enzyme-catalyzed reaction. The way to understand this is

KIA KB KC that C' is one less than the number of reactants in the enzyme-
ordered : v  catalyzed reaction plus one for enzymatic sites. The number of
mechanism < A> < A><B > < A><B><C> (30)
1    terms in the denominator of the rate equation (see column D) is
K IA KIA KB KIA KB KC
always equal to the number of steps in the mechanism. The sum
of the numbers of terms with single, double, and triple products
Vf < A><B ><C> of concentrations of reactants is always one less than D.
random KIA KB KC Ordered A + B P Mechanism
: v 
mechanism
1
< A> 
<B>  <C>  < A><B> The rate of the forward reaction is given by
K IA KIB KIC KIA KB
(31) Vf < A ><B>

< A ><C> 
<B ><C>
K IA K C KIB KC K IA KB
vf  (32)

< A ><B><C>
1
< A >  < A ><B>  <P >
KIA KB KC K IA K IA KB KP

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 Research: Science and Education

The denominator has been augmented by the [P]/KP term from called Haldane equations because he pointed out the first of
the rate equation for the reverse reaction. The rate of the reverse these relations in 1930. The mechanisms in Table 2 involve only
reaction is given by the following six Haldane equations:

Vr <P > Vf K P
K b S P
 (37)
Vr K S
KP (33)
vr 
1
<A> 
< A ><B>  <P > Vf KP
K IA K IA KB KP K b A + B P
 (38)
Vr KIA KB

The denominator has been augmented with [A]/KIA + [A][B]/ Vf KP K IQ

KIAKB from the rate equation for the forward reaction. The K b A + B P + Q
 (39)
rate equation for ordered A + B P is given by the difference Vr K IA KB
between eqs 32 and 33.
Vf KP
Vf < A ><B K b A + B + C P

>  Vr <P>
(40)
Vr K IA KB KC
K IA KB KP (34)
v 
1
< A>

< A ><B>

<P > Vf KP KIQ
K IA K IA KB KP K b A + B + C P + Q
 (41)
Vr KIA KB KC
This rate equation has been given in eq 12, but it is repeated here
so that it can be compared with other rate equations. Vf K P K IQ K R

K b A + B + C P + Q + R
 (42)
Random A + B P Mechanism Vr KIA KB KC

Vf < A ><B
>  Vr <P> Note that KIB, KIC, KIP, and KQ do not appear in these Haldane
K IA KB KP (35) equations. The Haldane equation gives a property of the reaction
v 
1
< A>

< B>

< A ><B >

<P> that is catalyzed, and it is independent of the mechanism.
K IA K IB K IA KB KP
Discussion
The denominator for the forward reaction has been Rapid-equilibrium rate equations are the ones to start with
augmented with [P]/KP and the denominator of the reverse in determining the mechanism of an enzyme-catalyzed reaction.
reaction has been augmented with 3 terms proportional to [A], If one of these rate equations represents all the experimental
[B], and [A][B]. Note that A and B play the same roles in this data, the Michaelis constants are equilibrium constants. If
rate equation. rapid-equilibrium rate equations do not have enough terms to
represent the experimental data, the complete steady-state rate
Ordered A + B Ordered P + Q Mechanism equation has to be used.
The values of the number of reactants N', the number of
Vf < A ><B > Vr <P><Q > components C', and the number of independent reactions R'
 for enzyme-catalyzed reactions can be calculated by adding the
K IA KB K P K IQ
v  (36) values in Table 1 for half-reactions. The number of reactants in
1
< A >  < A ><B>  <Q >  <P><Q >
K IA K IA KB K IQ K P K IQ
The terms in A and B play different roles, and so it is pos-
sible to determine whether A is bound first or second. This ap-
plies to the order of dissociation in the reverse reaction as well.
The steady-state rate equation has eleven denominator terms (4).
(The rate equations for the remaining mechanisms are provided
in the online supplement, along with an expanded Table 2.)
Haldane Equations
Rate equations that include the reverse reaction as well as
the forward reaction include the thermodynamics of the cata-
lyzed reaction as well as the kinetics for the two reactions. When
v = 0, the relation between the apparent equilibrium constant
K' and the kinetic parameters is obtained. These relations are
1140 Journal of Chemical Education  •  Vol. 85  No. 8  August 2008  •  www.JCE.DivCHED.org  •  © Division of Chemical Education
an enzyme-catalyzed reaction is equal to the sum of the N' of
the two half-reactions minus one so that E is not counted twice.
The number of components C' is determined by the catalyzed
reaction and is one less than the number of reactants in the
catalyzed reaction. The number of independent reactions R' is
one greater than the sum of the number of reactions in the two
half-reactions because the isomer interconversion is counted.
The sum of the number of single, double, and triple terms in the
denominator is one less than the number of denominator terms
because the enzyme site contributes the 1 in the denominator.
Note that in identifying N', C', and R', counting ES EP,
EAB EPQ, and EABC EPQR as one reactant (because
they are isomers) or as two reactants is optional. When both
members of the pair are counted as reactants (as is done in Table
2), there is one more reaction and one more reactant so that C'
is not affected. In this article both reactants and the isomeriza-
tion reaction are counted. This has the additional advantage that
 
 Research: Science and Education

when pH effects are discussed, the pH dependencies of Vf and
Vr can be different (8).
Complete rate equations provide a connection to thermo-
dynamics through the Haldane equation, and they can also be
used in calculating the way that systems of enzyme-catalyzed
reaction go from an initial state to equilibrium. However, kinetic
data discussed here cannot be used to unambiguously describe
the mechanism of a reaction. It is desirable to have additional
experimental results, isotope labeling, spectroscopic data, and
so forth to sort out alternative mechanisms.
Acknowledgments
2. Segel, I. H. Enzyme Kinetics: Behavoir and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems; Wiley: Hoboken,
NJ, 1975.
3. King, E. L.; Altman, C. J. Phys. Chem. 1956, 60, 1375–1378.
4. Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 104–137.
5. Roberts, D. V. Enzyme Kinetics; Cambridge University Press:
Cambridge, 1977.
6. Alberty, R. A. Biochemical Thermodynamics: Applications of Math-
ematica; Wiley: Hoboken, NJ, 2006.
7. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Portland
Press Ltd.: London, 2004.
8. Alberty, R. A. Biophys. Chem. 2006, 124, 11–17.

I am indebted to Robert N. Goldberg (NIST), Carl Frie- Supporting JCE Online Material
den (Washington University), and Athel Cornish-Bowden http://www.jce.divched.org/Journal/Issues/2008/Aug/abs1136.html
(France) for many helpful discussions. Grateful thanks to
Abstract and keywords
NIH for support of this research by award number 5-RO1-
GM48348-11. Full text (PDF)
Supplement
Literature Cited
The remaining mechanisms
1. Cha, S. J. Biol. Chem. 1968, 243, 820–825. Extended Table 2

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