Sungman Cha

and Steady State

or Combined Assumptions of Equilibrium
under the Rapid Equilibrium Assumption
Equations for Enzyme-catalyzed Reactions
A Simple Method for Derivation of Rate
ENZYMOLOGY:
1968, 243:820-825. J. Biol. Chem.

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THE JOURNAL cm BIOLOGICAL CHEMISTRY
Vol. 243, No. 4, Issue of February 25, pp. 820-825, 1968
hinted in U.S.A.
A Simple Method for Derivation of Rate Equations for
Enzyme-catalyzed Reactions under the Rapid
Equilibrium Assumption or Combined
Assumptions of Equilibrium and
Steadv State*
J
(Received for publication, July 28, 1967)
SUNGMAN CH.~
From the Division of Biological and Medical Sciences, Byown University, Providence, Rhode Island 02912
SUMMARY
A method is described by which rate equations for enzymic
reactions under the equilibrium assumptions may be derived
by inspecting the reaction pathways without solving simul-
taneous equations in the conventional way. Also described
is a simple graphic method which combines the above tech-
nique with King and Altmans method. By the latter method,
rate equations may be derived with minimal effort for com-
plicated enzymic mechanisms in which some steps are
considerably faster than the rest so that the enzymic species
connected by those rapid steps may be at a near equilibrium
while the over-all reaction is in a steady state. Rate equa-
tions are derived, for the purpose of illustration, for the
reversible single substrate-single modifier system, the
partial equilibrium ping-pong bi-bi mechanism, the general
mechanism for two-substrate system, and a complicated
three-substrate mechanism.
The method of King and Altman (1) has been successfully
used for the derivation of steady state rate equations for en-
zymic reactions, and the method can be further simplified by the
applicat,ion of the theory of graphs as shown by Volkenstein and
Goldstein (2). The steady state treatment of complicated reac-
tion mechanisms, however, leads to equations and constants so
complex that the basic kinetic properties of the mechanism may
be obscured. For this reason the equations derived on the basis
of rapid equilibrium assumptions are sometimes more useful
than the steady state equations. The purpose of this paper is
to describe a simplified method to derive rate equations under
* This study was supported in part, by United States Public
Health Service Research Grant GM 14093 from the National
Institute for General Medical Sciences.
the equilibrium assumptions or the combination of steady state
and equilibrium assumptions.
THEORY
When an enzyme-catalyzed reaction consists of more than one
step, and one or more portions of the whole reaction are signifi-
cantly faster than the over-all reaction, the partial reactions in
such segments must reach near equilibrium as the over-all reac-
tion reaches a steady state. A term, rapid equilibrium segment,
will be used to designate such a portion of the pathway which is
connected by one or more rapid steps without. including any
slow steps. In certain mechanisms, there may be only one rapid
equilibrium segment and all the enzyme species are included in
the segment. These special cases are known as rapid equilib-
rium mechanisms (3), and the simplest example of this kind is,
of course, the classic Michaelis-Menten mechanism (4). In
general, however, there may be some enzyme species which are
connected to others only through slow steps, or there may be two
or more rapid equilibrium segments isolated by one or more slow
steps. This latter case will be called in this paper the partial
equilibrium mechanism.
Another term, fractional concentration factor is defined for
the purpose of this paper as the fraction of the total enzyme in a
rapid equilibrium segment occurring as the species in question.
More specifically, the fractional concentration factor ( ji) for the
ith enzyme species (Ei) belonging to a rapid equilibrium segment
consisting of n enzyme species may be defined as
(1)
where Ni is the numerator of ji, and D, the denominator, is
common to the expressions of all ji, and D = C Ni since
Cji = 1.
Each numerator term Ni can be expressed in terms of the
820

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Issue of February 25, 1968 S. Cha 821
concentration variables (of ligands) and rate constants (k).
N; is the product of all rate constants and concentration variables
along the pathway from an arbitrarily chosen reference enzyme
species, El, to the ith enzyme species, Ei, in that direction, di-
vided by the product of all rate constants and concentration
variables in the reverse direction. The term for the reference
enzyme species, N1, is always 1. For all other enzyme species
the corresponding Ni term may be expressed as a fraction con-
sisting of the concentration variables and dissociation constants
or equilibrium constants. I f a step involves a binding of a
ligand to an enzyme species, the concentration variable (of the
ligand) appears in the numerator of Ni and the dissociation
constant in the denominator of Ni; but if the step involves a
dissociation, the concentration variable appears in the denomi-
nator and the dissociation constant in the numerator; and if the
step is unimolecular in both directions, the equilibrium constant
defined in the forward direction (from El to Ei) appears in the
numerator. When there is more than one pathway through
which El may be converted to Eiy any one and only one of these
pathways may be used for the evaluation of N i.l
Although the simplest form of enzyme, such as the free enzyme,
is usually chosen as the reference enzyme species (E1 in the
above), any enzyme species in the same rapid equilibrium seg-
ment may be chosen as the reference. For example, the frac-
tional concentration factors in a rapid equilibrium segment
kz hA
EB- EdEA --
kaB kz
(2)
may be evaluated by choosing E as the reference, thus
fE =
(E)
1
0-0 + @CA) + (EB) = l , h(A) I b(B)
kz ka
(3)
h(A)
(Ed kz
fEA = (E) + @A) + (EB) = 1, k,(A) I h(B)
kz ka
(4)
(4
K,
=
1+((A)+(&)
Ka Kh
h(B)
(EB) h
___ =
fEB = (E) + (EA) + (EB) k,(A)
1+7
+ !$E)
(5)
(B)
A
Kh
-
where K, and Kb are the dissociation constants of EA and EB,
respectively. Alternatively, EB may be chosen as the reference,
1 The proof of this rule is available on request.
then
Kh
fE = Kh(B) KhcAl =
1
(A) (B)
(6)
+(B)+(U)K, +K,+z
with the same result as before. Note that the step EB + Ed
involves the dissociation of B and the binding of A.
Derivation of Rate Equation for Rapid Equilibrium Mechanisms
-The over-all reaction velocity may be defined as the net flus
through the rate-limiting step (algebraic sum if alternative
pathways exist), and the derivation procedure reduces to the
evaluation of the fractional concentration factors of the species
involved in the rate-limiting steps, which can be done by in-
specting the reaction diagram as will be shown in the examples.
Derivaticm of Rate Equations for Partial Equilibrium Mech.-
anises-When there is at least one enzyme species or a rapid
equilibrium segment which is connected to other enzyme species
only by slow steps, the following method may be used to derive
the rate equation.
1. Draw a reaction pathway diagram that includes all the
enzyme species and the interconversion steps with the rate
constants and concentration terms, e.g. (S) and (P).
2. Assign a new symbol to each rapid equilibrium segment,
X, X, etc. For each slow step by which any enzyme species in
the rapid equilibrium segment (X) is converted to other species
outside the segment, multiply the rate constant (ki) for the step
by a fractional concentration factor ( fi), so that the rate of flux
through this step can be represented as kifi(X), where kifi is an
apparent rate constant. For reaction in the reverse direction
along this step, the rate constant is unchanged, unless the step
connects two rapid equilibrium segments, in which case both
rate constants are multiplied by their respective fractional con-
centration factors. Draw a new reaction pathway diagram
employing new symbols.
3. Apply King and Altmans method to the above newly
constructed reaction scheme. Regard each rapid equilibrium
segment as if it were an enzyme species, and use the apparent
rate constants, e.g. fikl, in place of the rate constant, kl.
4. Finally, the fractional concentration factors expressed in
terms of concentrations and dissociation constants are substituted
into the equation derived in the previous step.
EXAMPLES
Example 1. Reversible Single Substrate-Single MoclifLer Mechanism
Rapid Equilibrium Case-The single substrate-single modifier
mechanism has already attracted the attention of many kinet-
ic&s, among whom are Segal, Kachmar, and Boyer (5), Botts
and Morales (6), King (7), Ari&ns, van Rossum, and Simonis
(8), and Frieden (9). The mechanism involves six enzyme-
containing species and nine interconversion steps
E+S
h
-----ES KG = kz/kl (7)
kz
ka
ES-----EP
kg
Ksi, = ks/k4 (8)
ks
El--E+P
kg
Keg = k&s (9)

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s22 Derivation of Enzyme Rate Equations Vol. 243, No. 4
h
E+M- - ME
Its
ES+ M
ks
- MES
k 10
EP+M
hl
- MEP
k 12
Kno = b/k? (10)
converted to the other species, any pathway may be used to
represent the fractional concentration of the latter species. For
instance in the above equation, (MES)/(E,) was evaluated by
Km8 = km/b (11)
the pathway, E + ME -+ MES, but it could be done by E --f
ES --t MES as well, then the numerator term of the fraction
becomes (8) (M) /&;KK;;~~ instead of (M) (8) /KmoKm;. But
Kmp = knlku (1% these two apparently different terms are actually identical be-
cause K,,K;;;, = K;,K,;.
ME+ S
k
----% MES
k
14
k
MES 15 MEP
k 16
ku
MEP--ME+P
km
E + B
h
w EB
k9
k
where S is the substrate, P is the product, and M is the modifier.
p,EQ&
E+Q (20)
ks
k
13 hz
Also shown are the definitions of the dissociation constant or the
equilibrium constant of each step.
suppose two steps, EA = EP and EB = EQ, are much slower,
As repeatedly pointed out by the above mentioned authors,
compared to the rest, then the reaction may be represented by
Kms = ku/kn (1% Example 2. Partial Equilibrium Ping-Pung Bi-Bi Mechanism
Simple Case with Two Rapid Equilibrium Segments-In the
Km,, = ks/kle (14)
ping-pong bi-bi mechanism
E-I-A
h k,
- EA -
ks
- -
EP - E + P (19)
K,i, = kn/kls (15)
h kd -x-
the steady state treatment of this mechanism leads to a rate
equation too complicated to be of much practical value. How-
ever, if assumptions are made that the isomerization steps,
ES e EP and MES s MEP, are the slow ones, and all other
steps are in rapid equilibrium, the rate equation may be written
down by inspecting the following reaction pathway diagram
without solving any simultaneous equations.
i . . . . . . . . . . . . . . . . . . . i (slow) : .
DIAGRAM II
4A
DIAGRAM I
k,oXo
DIAGRAM IIa
Since the velocity may be expressed as the net flux through
the rate-limiting steps,
or
o = k,(ES) + ku(MES) - kd(EP) - k&fEP) (16)
v = [ksja + kujn - kJa - k&lUL) (17)
where h fib, f4, and fi6 represent (ES) l(EJ, (MES) l(&),
(EP) /(E,) and (MEP) /(EJ, respectively. Expressing these
factors in terms of concentration variables and dissociation con-
stants
V= (18)
Note that the denominator of the above equation is the sum of
the numerators of all the fractional concentration factors.
The first term 1 represents E, and the rest represent ES, ME,
MES (via ME), MEP (via ME), and EP, respectively, in that
order. As mentioned above, when there are alternative path-
ways through which the reference species (E in this case) may be
X
Diagram II or IIa where X and X represent the rapid equilib-
rium segments, EA = E ti EQ, and EP + E G EB, respec-
tively, and
LX) = (El + @A) + (E&l (21)
(X) = (E) + (EB) + (EP) (22)
and the fractional concentration factors may be expressed as
k,(A)
WA)
f3=(x)=
kz
MA)
lf-
k&Q)
k) +k 11
= k2kn + klkn(A) + k&la(Q)
ka(P)
(23)
(EP)
f4 = (xl) =
ks
h(B)
1+-
k@)
ks +ks
k&,(P)
= kck, + ksk,(B) + k&&Y
(24)

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Issue of February 25, 1968 S. Cha
k,(B)
(EB)
=ix.)=1+
Its
k,(B) kdp)
k+k-
8 a
k&(B)
= k&g + kjk,(B) + k6kdP)
(EQ)
h(Q)
f10 = 0 = 1 +
kn
k,(A) kd&)
kl+T-
11
k&d&)
(25)
E\ AGXY\ HE
EGX EGY
\/
EG
DIAGRAM I II
(26) For the purpose of this paper, this mechanism may be repre-
sented by Diagram IIIa. The derivation of full steady state
= ktkll + klkn(A) + kzkd&)
~. ._ ._. ___ .___ .._.
Xi
EB EP
An application of King and Altmans method to Diagram IIa
yields
(27)
823
Substituting Equations 23 to 26 into Equation 27, and rearrang-
ing
(E,)[k,k,ksk,k,k,,(A) (B) - kzk4ksksk,okdP)(Q)l
= k&k5kskll(A) + ktksk,kskn(B)
+ klksk,ktdka + ks)(A)(B) + kzk,k,kskll(P)
+ kzkSk8k10kdQ) + k3k6ksk12@+ kd (P) (&I
(28)
+ k&&&n &a + kd (A) (PI
+ k?kskTklz(ks + kd (B) (Q)
This equation may be compared to EquaCon 29 derived by the
total steady state treatment of the same mechanism.
(EJ[k,ksk,k,ksk,,(A)(B) - k,kaksksklokdP)(Q)l
= kJiaka(kska + k*kll + kskd(A)
+k,ks kn(kzkr + ktks + kzkd(B)
+ [klk,kgkll(ka + k, + k5) + klkakekT(ks + klo + kdl
. (A)(B) + kzkaks(ksho + k&n + k&d U)
(29)
+ ksklOklz(kzkc + kzks + kakd(Q) + [kskak1okdkz + ka
+ kd) + kzkakskln(ks + ks + kd](P)(Q) + klkdk3
+ kd)(kaklo + kskll + k&d(A)(P) + kddks
+ klo) (kzk, + k,ks + k3ks) (B) (Q)
It can be easily shown that Equation 29 reduces to Equation 28,
as it should, when kS, kd, ks, and klo are made much smaller than
the other unimolecular rate constants, kz, kj, ks, and kll.
Example 3. General Mechanism for Two-Substrate Systems
Case of Rate-limiting Step within a Rapid Equilibrium Segment
-Wang and Hanes (10) pointed out that there are 784 terms,
including cubic terms in some of the concentration variables, in
the full steady state rate equation for the following mechanism,
which they considered to be the general mechanism for two-
substrate systems.
: .._.. .._..
kr
4
. . . . ..-.-.........~..-----........-..............-.---.. ........--......
k,
ks
....--.....---.._...
Xi K:, Mb
~....----........--.....-..-
EP- - E - EB
DIAGRAM IIIa
rate equation for this mechanism will be extremely difficult
for obvious reasons. Partial equilibrium assumptions, however,
simplify the equation. Assume that the binding and dissocia-
tion of the reaction components are much faster than the actual
group transfer reaction involving covalent linkages, then there
are three slow steps indicated by the single-lined double-headed
arrows in Diagram IIIa, while the rest of the steps are assumed
to be in rapid equilibrium. Now the reaction mechanism may
be represented by
kf,
krfi
k,fi
K-2
kzf, X
X
ksfs
ksfs
DIAGRAM IIIb
where X and X represent the two rapid equilibrium segments so
that
(X) = (E) + (EA) + (EB) + (EAB)
+ (EP) + (EQ) + @P&l
(30)
(X) = (E) + (EB) + (EP)
(31)
and the fractional concentration fact.ors are defined and evaluated
as follows
64) (B)
WAN
f1=(,)=
K&
1+!$+g+Gg
(3%
$0 21,
+ (;I I (Q) I (P)(Q)
Ki, Ki, K&i,

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824 Derivation of Enzyme Rate Equations Vol. 243, No. 4
(P)(Q)
K,Ki,
V-G)
h(A) (Bj k&3 (Q)
>(
h(B) k,(P)
~ - K,Ki,
__ __
(EPQ)
fi = (Xl
KiaKb K;a + K:,
(A) u-3) (A)(B)
l+jy+K+-
&a&
(33) +
k,ks(A) (B) kd&) (&I
za Lb
+ (p) + 9 + P)(Q)
v=
Ki,Kla - K&Ki,
,1
K, K, f&Ki,
l I (A) I (B) + (A) U3 I 09 I (Q) I (P)(Q)
Ki, Kib K,,Kb Ki, Kc K,Ki,
(43)
f
ks(B) k,(P)\
-I-
(-4
\ K:b T K:, /
(E-4
fa=(x)=
Ki,
1++@+(A)(B)
+ 1 + (B) + (p)
(34)
>(
k&I) k.(Q)
-+-
Kib K:, Ki, Ki,
Ki, Kia &a&
+ (p) + (&) + 0) (&I
It should be pointed out that the part of velocity due to the path-
Ki, Ki, K&q
way through E can be taken as k5(EB) - kc(EQ) instead of
k,(EA) - kd(EP).
(P)
Also note that, when the flux through E is negligible, i.e.
-
f Ki,
k3 = k4 = ks = k6 = 0, Equation 38 reduces to that of the rapid
1
= @PI
equilibrium random bi-bi mechanism (3); and that, when the
(X)
(35)
flux through the central complex, EAB = EPQ, is negligible,
i.e. Kb = Kib = K, = Kip = m, the equation reduces to that
of Example 2 above.
(B)
f. = (EH
Kib
Example 4. Example of Mechanism with Enzyme Species Not
0
- =
(Xj 1 I (B) I (P)
(36)
Included in Rapid Equilibrium Segment
Kit, _xp
Another mechanism, for which the rate equation has been
derived by Cleland2 will be used for a further illustration. In
(&I
this example, one of the four slow steps (E = EA) connects two
- enzyme species, one of which does not belong to any rapid equi-
f6 =
(E&j Kiq
-=
(Xj (A) (B) (A) (B)
+ K, + K;, + K;,Kh
(37)
.1 . .-
(PI (Q) 03 C&j
L-L-L-
For Diagram IIIb, there are only two King and Altman patterns
xwx x
which yield
tm k4f4 + k5f5
-=
(Ed
k,j3 + kdf4 + ksfb + kcf6
(38)
(-m kafa + kGf6
-=
BJ k3fz + k4f4 + ksfs + kefe
(39) DIaGRlM Iv
Note that the above two patterns do not include the closed loop
librium segment. In Diagram IV where the single-lined arrows
beginning and ending in the same rapid equilibrium segment
represent the slow steps, the double-lined arrows the rapid steps,
(X) in the left-hand loop of Diagram IIIb.
and K1, K2, etc., are the dissociation constants. Two rapid
The partial steady state rate may be set as
equilibrium segments, X and X, are indicated by broken-lined
rectangles. Now the mechanism may be represented by
0 = kl(EAB) - k?(EPQ) + kz(EA) - kd(EP) F (40)
2r = k,fi(Xj - k&(X) + kafdX) - kdfa(X)
.(--E-y
(41)
2
if,
i
Substituting Equations 38 and 39 into Equation 41,
or
(E,)[(klfi - kpfz)(k,f, + ksfd + (kafakbfs - bf&fdl
v=
kzf3 + kdf4 + ksfs + kcfe
(42)
DIAGRAM IVa
i 1
.._._......_____.................................. __....____......___:
: \
\
Ic3
E + products
Substituting the values off (Equations 32 to 37) into Equation 42 * W. W. Cleland, personal commumcatron.

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Issue of February 25, 1968 S. Cha 825
An application of King and Altmans method to this scheme gives
63 k&b f , + k, f-h, f7 + ka f&, f7
_=_
(-Ed c
(44)
m kl (A) Wi
(E,)=c
(45)
(X) h(A)hjs
-=
U-L)
c
(46)
where the denominator, c is the sum of all three numerators.
The velocity may be expressed as
ZJ = ks(EABC) + k,(FBC) = kjj,(X) + kTj,(X)
Substituting Equations 45 and 46 into Equation 47
kkNk& + ksfdk&(Eo)
' = h(A)(k& + kifd + k,f,(krf? + k& + ksjd
Substituting the following three equations
WA)
fz = f3 = (x) =
(B) (3
(EABC)
f5=(x)=
KtK,
m ((3
l+K,+Kn+
(B) (Cl
KJG
(B)(C)
(FBC)
f6 = (x, =
KZKT
l + (B) + (c) + (B)(C)
KS Ks KSKT
into Equation 48, the rate equation becomes
v=
k,(A)(B)(C)
KsK1.
which may be expressed differently by
ploging the relationship, k&, = K&g
(Cl (B) ((3
Tg+KlKa
)I
k3) +
k&3 (0
yjy-
1 3
1
(47)
(48)
(49)
(50)
(51)
(52)
rearranging and em-
v x
La
n
(53)
7
1 + (B) + (c) + (B)(C) +(h + kd + kdB)(C)
Kl K2 -->
KlKs k,(A) h(A)K&
9.
10.
11.
12.
13.
DISCUSSION
The equilibrium rate equation, although it is only an approxi-
mation of the true kinetics, is simpler and more convenient to use
than the steady state equation, and it can offer valuable insight
into the reaction mechanism. The derivation of equilibrium
rate equations by the conventional method amounts to solving
as many simultaneous linear equations as the number of enzyme
species, and the algebraic manipulation can be very cumbersome
when there are many enzyme species. It is hoped that the use
of the simple rules described in this paper may contribute to the
derivation and the analysis of complicated enzymic mechanisms
such as kinetics of regulatory enzymes. Although considerable
progress has been made by investigators, notably by Monod,
Wyman, and Changeux (11) and Koshland, Nbmethy, and Filmer
(12) as reviewed by Atkinson (13), much more is desired to be
done.
Rate equations for partial equilibrium mechanisms, of course,
can be derived by imposing appropriate restrictions on the full
steady state equations, as shown in Example 2. However,
use of the present graphic method, which is a logical hybrid of
the equilibrium treatment and King and Altmans schematic
method for the steady state, saves a considerable effort for the
derivation. With the present method and King and Altmans
method or that of Volkenstein and Goldstein, it is now possible
to derive rate equations for almost any enzyme mechanism under
various assumptions through a simple inspection of the reaction
pathway or by a simple schematic method, without solving
simultaneous equations.
Acknozoledgnzents-I wish to thank Professor R. E. Parks, Jr.,
and Professor W. W. Cleland for their encouragement and
advice.
1.
2.
3.
4.
5.
6.
7.
8.
REFERENCES
KING, E. L., AND ALTMAN, C., J. Phys. Chem., 60,1375 (1956).
VOLKENSTEIN, M. V., AND GOLDSTEIN, B. N., Biochem. Biophys.
Acta, 116, 471 (1966).
CLELAND, W. W., Biochem. Biophys. Acta, 67, 104 (1963).
MICHAELIS, L., AND MENTEN, M. L., Biochem. Z., 49, 333
(1913).
SEGAL, H. L., KACH~~AR, J. R., AXD BOYER, P. D., Enzymologia,
16, 187 (1952).
BOTTS, J., AND MOR.LLES, J. F., Trans. Faraday Sot., 49, 696
(1953).
KING, E. L., J. Phys. Chem., 60, 1378 (1956).
ARI~NS, E. J., VAN ROSSUM, J. M., AND SIMONIS, A. M.,
ArzneimitteZ-Forschung, 6, 611 (1956).
FRIEDEN, C., J. Biol. Chem., 239, 3522 (1964).
WONG, J. T.-F., AND HANES, C. S., Can. J. Biochem. Physiol.,
40, 763 (1962).
MONOD. J.. WYMAN. J., .ZND CHINGEUX, J. P., J. Mol. Biol..
12, 8s (1965).
KOSHLAND, D. E., JR., ??~METHY, G., AND FILMER, D., Bio-
chemistry, 6, 365 (1966).
ATKINSON, D. E., Annu. IZeo. Biochem., 36, 85 (1966).

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