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

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SUMMARY the equilibrium assumptions or the combination of steady state
A method is described by which rate equations for enzymic and equilibrium assumptions.
reactions under the equilibrium assumptions may be derived
THEORY
by inspecting the reaction pathways without solving simul-
taneous equations in the conventional way. Also described When an enzyme-catalyzed reaction consists of more than one
is a simple graphic method which combines the above tech- step, and one or more portions of the whole reaction are signifi-
nique with King and Altman’s method. By the latter method, cantly faster than the over-all reaction, the partial reactions in
rate equations may be derived with minimal effort for com- such segments must reach near equilibrium as the over-all reac-
plicated enzymic mechanisms in which some steps are tion reaches a steady state. A term, rapid equilibrium segment,
considerably faster than the rest so that the enzymic species will be used to designate such a portion of the pathway which is
connected by those rapid steps may be at a near equilibrium connected by one or more rapid steps without. including any
while the over-all reaction is in a steady state. Rate equa- slow steps. In certain mechanisms, there may be only one rapid
tions are derived, for the purpose of illustration, for the equilibrium segment and all the enzyme species are included in
reversible single substrate-single modifier system, the the segment. These special cases are known as rapid equilib-
partial equilibrium ping-pong bi-bi mechanism, the general rium mechanisms (3), and the simplest example of this kind is,
mechanism for two-substrate system, and a complicated of course, the classic Michaelis-Menten mechanism (4). In
three-substrate mechanism. 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 method of King and Altman (1) has been successfully the purpose of this paper as the fraction of the total enzyme in a
used for the derivation of steady state rate equations for en- rapid equilibrium segment occurring as the species in question.
zymic reactions, and the method can be further simplified by the More specifically, the fractional concentration factor ( ji) for the
applicat,ion of the theory of graphs as shown by Volkenstein and ith enzyme species (Ei) belonging to a rapid equilibrium segment
Goldstein (2). The steady state treatment of complicated reac- consisting of n enzyme species may be defined as
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 (1)
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 where Ni is the numerator of ji, and D, the denominator, is
common to the expressions of all ji, and D = C Ni since
* This study was supported in part, by United States Public
Health Service Research Grant GM 14093 from the National Cji = 1.
Institute for General Medical Sciences. Each numerator term Ni can be expressed in terms of the

820
Issue of February 25, 1968 S. Cha 821

concentration variables (of ligands) and rate constants (k). then

N; is the product of all rate constants and concentration variables
Kh
along the pathway from an arbitrarily chosen reference enzyme
1
species, El, to the ith enzyme species, Ei, in that direction, di- fE = Kh(B) KhcAl = (6)
(A) (B)
vided by the product of all rate constants and concentration
variables in the reverse direction. The term for the reference ‘+(B)+(U)K, ‘+K,+z
enzyme species, N1, is always 1. For all other enzyme species with the same result as before. Note that the step EB + Ed
the corresponding Ni term may be expressed as a fraction con-
involves the dissociation of B and the binding of A.
sisting of the concentration variables and dissociation constants Derivation of Rate Equation for Rapid Equilibrium Mechanisms
or equilibrium constants. If a step involves a binding of a
-The over-all reaction velocity may be defined as the net flus
ligand to an enzyme species, the concentration variable (of the through the rate-limiting step (algebraic sum if alternative
ligand) appears in the numerator of Ni and the dissociation pathways exist), and the derivation procedure reduces to the
constant in the denominator of Ni; but if the step involves a
evaluation of the fractional concentration factors of the species
dissociation, the concentration variable appears in the denomi- involved in the rate-limiting steps, which can be done by in-
nator and the dissociation constant in the numerator; and if the
specting the reaction diagram as will be shown in the examples.
step is unimolecular in both directions, the equilibrium constant Derivaticm of Rate Equations for Partial Equilibrium Mech.-
defined in the forward direction (from El to Ei) appears in the
anises-When there is at least one enzyme species or a rapid
numerator. When there is more than one pathway through equilibrium segment which is connected to other enzyme species
which El may be converted to Eiy any one and only one of these only by slow steps, the following method may be used to derive
pathways may be used for the evaluation of N i.l the rate equation.
Although the simplest form of enzyme, such as the free enzyme, 1. Draw a reaction pathway diagram that includes all the

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is usually chosen as the reference enzyme species (E1 in the enzyme species and the interconversion steps with the rate
above), any enzyme species in the same rapid equilibrium seg- constants and concentration terms, e.g. (S) and (P).
ment may be chosen as the reference. For example, the frac-
2. Assign a new symbol to each rapid equilibrium segment,
tional concentration factors in a rapid equilibrium segment X, X’, etc. For each slow step by which any enzyme species in
kz hA the rapid equilibrium segment (X) is converted to other species
EB- -- EdEA (2) outside the segment, multiply the rate constant (ki) for the step
kaB kz
by a fractional concentration factor ( fi), so that the rate of flux
may be evaluated by choosing E as the reference, thus through this step can be represented as kifi(X), where kifi is an
(E) 1 apparent rate constant. For reaction in the reverse direction
fE = along this step, the rate constant is unchanged, unless the step
0-0 + @CA) + (EB) = l , h(A) I b(B)
connects two rapid equilibrium segments, in which case both
kz ka
(3) rate constants are multiplied by their respective fractional con-
centration factors. Draw a new reaction pathway diagram
employing new symbols.
3. Apply King and Altman’s method to the above newly
constructed reaction scheme. Regard each rapid equilibrium
h(A) segment as if it were an enzyme species, and use the apparent
(Ed kz rate constants, e.g. fikl, in place of the rate constant, kl.
fEA = (E) + @A) + (EB) = 1, k,(A) I h(B) 4. Finally, the fractional concentration factors expressed in
kz ka terms of concentrations and dissociation constants are substituted
(4) into the equation derived in the previous step.
(4
K, EXAMPLES
=
1+((A)+(&) Example 1. Reversible Single Substrate-Single MoclifLer Mechanism
Ka Kh
Rapid Equilibrium Case-The single substrate-single modifier
h(B) mechanism has already attracted the attention of many kinet-
(EB) ___ = h ic&s, among whom are Segal, Kachmar, and Boyer (5), Botts
fEB = (E) + (EA) + (EB) k,(A) + !$E) and Morales (6), King (7), Ari&ns, van Rossum, and Simonis
1+7
(8), and Frieden (9). The mechanism involves six enzyme-
(5) containing species and nine interconversion steps
(B)
A
h
- Kh
E+S -----‘ES KG = kz/kl (7)
kz
ka
ES-----‘EP Ksi, = ks/k4 (8)
where K, and Kb are the dissociation constants of EA and EB, kg
respectively. Alternatively, EB may be chosen as the reference, ks
El’--E+P Keg = k&s (9)
1 The proof of this rule is available on request. kg
s22 Derivation of Enzyme Rate Equations Vol. 243, No. 4

h converted to the other species, any pathway may be used to

E+M- - ME Kno = b/k? (10)
Its represent the fractional concentration of the latter species. For
instance in the above equation, (MES)/(E,) was evaluated by
ks the pathway, E + ME -+ MES, but it could be done by E --f
ES+ M - MES Km8 = km/b (11)
‘k 10 ES --t MES as well, then the numerator term of the fraction
becomes (8) (M) /&;KK;;~~ instead of (M) (8) /KmoKm;. But
hl
EP+M - MEP Kmp = knlku (1% these two apparently different terms are actually identical be-
k 12 cause K,,K;;;, = K;,K,;.
k
ME+ S ----% MES Kms = ku/kn (1% Example 2. Partial Equilibrium Ping-Pung Bi-Bi Mechanism
‘k 14
Simple Casewith Two Rapid Equilibrium Segments-In the
k ping-pong bi-bi mechanism
MES 15 MEP Km,, = ks/kle (14)
‘k 16
h k, ks
E-I-A -- EA -- E’P - E’ + P (19)
ku h kd -x-
MEP--ME+P K,i, = kn/kls (15)
km h k9 k
E’ + B w E’B p,EQ& E+Q (20)
where S is the substrate, P is the product, and M is the modifier. ks k 13 hz
Also shown are the definitions of the dissociation constant or the
equilibrium constant of each step. suppose two steps, EA = E’P and E’B = EQ, are much slower,
As repeatedly pointed out by the above mentioned authors, compared to the rest, then the reaction may be represented by
the steady state treatment of this mechanism leads to a rate

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equation too complicated to be of much practical value. How- X’
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

k,oXo
DIAGRAM I DIAGRAM IIa
Diagram II or IIa where X and X’ represent the rapid equilib-
Since the velocity may be expressed as the net flux through
rium segments, EA = E ti EQ, and E’P + E’ G E’B, respec-
the rate-limiting steps,
tively, and
o = k,(ES) + ku(MES) - kd(EP) - k&fEP) (16)
LX) = (El + @A) + (E&l (21)
or (X’) = (E’) + (E’B) + (E’P) (22)
v = [ksja + kujn - kJa - k&lUL) (17)
and the fractional concentration factors may be expressed as
where h fib, f4, and fi6 represent (ES) l(EJ, (MES) l(&), k,(A)
(EP) /(E,) and (MEP) /(EJ, respectively. Expressing these
WA) kz
factors in terms of concentration variables and dissociation con- f3=(x)=
stants MA) k&Q)
lf- (23)
k) +k 11

V= (18) = k2kn + klkn(A) + k&la(Q)

ka(P)
Note that the denominator of the above equation is the sum of (E’P) ks
f4 = (xl) = h(B) k@)
the numerators of all the fractional concentration factors.
1+- ks +ks (24)
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- k&,(P)
ways through which the reference species (E in this case) may be = kck, + ksk,(B) + k&&Y
Issue of February 25, 1968 S. Cha 823

k,(B)
(E’B) Its
k,(B) kdp)

E\ AGXY\ HE
“=ix.)=1+ k+k-8 (25)
a

k&(B) EGX EGY

= k&g + kjk,(B) + k6kdP)
\/
h(Q) EG
(EQ) kn DIAGRAM I II
k,(A) kd&)
f10 = 0 = 1 + (26) For the purpose of this paper, this mechanism may be repre-
kl+T- 11
sented by Diagram IIIa. The derivation of full steady state
k&d&) ~. ._._. ___.___.._.
= ktkll + klkn(A) + kzkd&) Xi EB EP

An application of King and Altman’s method to Diagram IIa

yields

(27)

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: .._.. .._..
Substituting Equations 23 to 26 into Equation 27, and rearrang- kr 4 k, ks
ing X’i ......-.-.........~..-----........-..............-.---..
K:, ........--......
Mb....--.....---.._...
~....----........--.....-..-
E’P- E’ - E’B
(E,)[k,k,ksk,k,k,,(A) (B) - kzk4ksksk,okdP)(Q)l DIAGRAM IIIa
’ = k&k5kskll(A) + ktksk,kskn(B)

+ klksk,ktdka + ks)(A)(B) + kzk,k,kskll(P) rate equation for this mechanism will be extremely difficult
(28) for obvious reasons. Partial equilibrium assumptions, however,
+ kzkSk8k10kdQ) + k3k6ksk12@+ kd (P) (&I simplify the equation. Assume that the binding and dissocia-
+ k&&&n &a + kd (A) (PI tion of the reaction components are much faster than the actual
group transfer reaction involving covalent linkages, then there
+ k?kskTklz(ks + kd (B) (Q)
are three slow steps indicated by t’he single-lined double-headed
This equation may be compared to EquaCon 29 derived by the arrows in Diagram IIIa, while the rest of the steps are assumed
total steady state treatment of the same mechanism. to be in rapid equilibrium. Now the reaction mechanism may
be represented by
(EJ[k,ksk,k,ksk,,(A)(B) - k,kaksksklokdP)(Q)l
kf,
’ = kJiaka(kska + k*kll + kskd(A)
+k,ks kn(kzkr + ktks + kzkd(B) krfi
+ [klk,kgkll(ka + k, + k5) + klkakekT(ks + klo + kdl k,fi kzf, X X’
. (A)(B) + kzkaks(ksho + k&n + k&d U’) K-2 ksfs
(29)
ksfs
+ ksklOklz(kzkc + kzks + kakd(Q) + [kskak1okdkz + ka
DIAGRAM IIIb
+ kd) + kzkakskln(ks + ks + kd](P)(Q) + klkdk3
where X and X’ represent the two rapid equilibrium segments so
+ kd)(kaklo + kskll + k&d(A)(P) + kddks
that
+ klo) (kzk, + k,ks + k3ks) (B) (Q)
(X) = (E) + (EA) + (EB) + (EAB)
(30)
It can be easily shown that Equation 29 reduces to Equation 28, + (EP) + (EQ) + @P&l
as it should, when kS, kd, ks, and klo are made much smaller than (X’) = (E’) + (E’B) + (E’P) (31)
the other unimolecular rate constants, kz, kj, ks, and kll.
and the fractional concentration fact.ors are defined and evaluated
Example 3. GeneralMechanism for Two-Substrate Systems as follows
Case of Rate-limiting Step within a Rapid Equilibrium Segment 64) (B)
-Wang and Hanes (10) pointed out that there are 784 terms, WAN K&
f1=(,)=
including cubic terms in some of the concentration variables, in 1+!$+g+Gg
the full steady state rate equation for the following mechanism, (3%
$0 21,
which they considered to be the general mechanism for two- + (;I I (Q) I (P)(Q)
substrate systems. Ki, Ki, K&i,
824 Derivation of Enzyme Rate Equations Vol. 243, No. 4

(EPQ)
(P)(Q)
V-G) h(A)
~ KiaKb
(Bj k&3(Q)
- K,Ki,
h(B)
__
K;a +
k,(P)
__
K:,
K,Ki, >(
fi = (Xl (A)
l+jy+K+-
za
u-3)
Lb
(A)(B)
&a&
+ (p) + 9 + P)(Q)
(33)
v= l I (A) I (B) + (A) U3 I 09
+
k,ks(A)
Ki,Kla
(B)
-
kd&)
K&Ki,
I (Q) I (P)(Q)
(&I
,1
(43)
K, K, f&Ki, Ki, Kib K,,Kb Ki, Kc K,Ki,
f ks(B)
-I- k,(P)\
(-4 \ K:b T K:, /
(E-4 Ki, k&I)
+ 1+ (B)+ (p) -+- k.(Q)
K:, >( Ki,
fa=(x)=
1+‘“‘+@+(A)(B) Kib Ki,
(34)
Ki, Kia &a& It should be pointed out that the part of velocity due to the path-
+ (p) + (&) + 0’) (&I way through E’ can be taken as k5(E’B) - kc(EQ) instead of
Ki, Ki, K&q k,(EA) - kd(E’P).
Also note that, when the flux through E’ is negligible, i.e.
-(P) k3 = k4 = ks = k6 = 0, Equation 38 reduces to that of the rapid
f = @‘PI Ki, equilibrium random bi-bi mechanism (3); and that, when the
1 (35)
(X’) 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)
Example 4. Example of Mechanism with Enzyme Species Not

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f. 0 = -(E’H Kib Included in Rapid Equilibrium Segment
= (36)
(X’j 1 I (B) I (P)
Another mechanism, for which the rate equation has been
Kit, _xp
derived by Cleland2 will be used for a further illustration. In
this example, one of the four slow steps (E = EA) connects two
-(&I enzyme species, one of which does not belong to any rapid equi-
(E&j Kiq
f6 = -=
(Xj (A) (B) (A) (B)
(37)
’ + K, .1 + K;, . + .-
K;,Kh
(PI
L-L-L- (Q) 03 C&j

For Diagram IIIb, there are only two King and Altman patterns
i 1 .._._......_____..................................
__....____......___:
: \
\
xwx’ x Ic3
E + products

which yield
tm
-=
k4f4 + k5f5
(38)
(Ed k,j3 + kdf4 + ksfb + kcf6

(-m kafa + kGf6

-= (39) DIaGRlM Iv
BJ k3fz + k4f4 + ksfs + kefe
librium segment. In Diagram IV where the single-lined arrows
Note that the above two patterns do not include the closed loop
represent the slow steps, the double-lined arrows the rapid steps,
beginning and ending in the same rapid equilibrium segment
and K1, K2, etc., are the dissociation constants. Two rapid
(X) in the left-hand loop of Diagram IIIb. equilibrium segments, X and X’, are indicated by broken-lined
The partial steady state rate may be set as Now the mechanism may be represented by
rectangles.
0 = kl(EAB) - k?(EPQ) + kz(EA) - kd(E’P) F (40)

or .(--E-y
Substituting
2r = k,fi(Xj

Equations
- k&(X) + kafdX)

38 and 39 into Equation

- kdfa(X’)

41,
(41)
2 if, i
(E,)[(klfi - kpfz)(k,f, + ksfd + (kafakbfs - bf&fdl (42)
v=
kzf3 + kdf4 + ksfs + kcfe
DIAGRAM IVa
Substituting the values off (Equations 32 to 37) into Equation 42 * W. W. Cleland, personal commumcatron.
Issue of February 25, 1968 S. Cha 825

An application of King and Altman’s method to this scheme gives DISCUSSION

63
_=_ k&b f, + k, f-h, f7 + kaf&, f7 The equilibrium rate equation, although it is only an approxi-
(44)
(-Ed c mation of the true kinetics, is simpler and more convenient to use
m kl (A) Wi than the steady state equation, and it can offer valuable insight
(45) into the reaction mechanism. The derivation of equilibrium
(E,)=c
rate equations by the conventional method amounts to solving
(X’) h(A)hjs as many simultaneous linear equations as the number of enzyme
-= (46)
U-L) c species, and the algebraic manipulation can be very cumbersome
when there are many enzyme species. It is hoped that the use
where the denominator, c is the sum of all t’hree numerators.
of the simple rules described in this paper may contribute to the
The velocity may be expressed as
derivation and the analysis of complicated enzymic mechanisms
ZJ = ks(EABC) + k,(FBC) = kjj,(X) + kTj,(X’) (47) such as kinetics of regulatory enzymes. Although considerable
Substituting Equations 45 and 46 into Equation 47 progress has been made by investigators, notably by Monod,
kkNk& + ksfdk&(Eo) Wyman, and Changeux (11) and Koshland, Nbmethy, and Filmer
(48) (12) as reviewed by Atkinson (13), much more is desired to be
' = h(A)(k& + kifd + k,f,(krf?+ k& + ksjd done.
Substituting the following three equations Rate equations for partial equilibrium mechanisms, of course,
can be derived by imposing appropriate restrictions on the full
fz = f3 = WA)
(x) = steady state equations, as shown in “Example 2.” However,
(49)
use of the present graphic method, which is a logical hybrid of
the equilibrium treatment and King and Altman’s schematic

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(B) (‘3 method for the steady state, saves a considerable effort for the
(EABC) KtK, derivation. With the present method and King and Altman’s
f5=(x)= (50) method or that of Volkenstein and Goldstein, it is now possible
m ((3 (B) (Cl
l+K,+Kn+ KJG to derive rate equations for almost any enzyme mechanism under
various assumptions through a simple inspection of the reaction
(B)(C) pathway or by a simple schematic method, without solving
(FBC) KZKT simultaneous equations.
f6 = (x, = (51)
l + (B) + (c) + (B)(C)
KS Ks KSKT Acknozoledgnzents-I wish to thank Professor R. E. Parks, Jr.,
and Professor W. W. Cleland for their encouragement and
into Equation 48, the rate equation becomes advice.
k,(A)(B)(C) REFERENCES
KsK1.
v= 1. KING, E. L., AND ALTMAN, C., J. Phys. Chem., 60,1375 (1956).
2. VOLKENSTEIN, M. V., AND GOLDSTEIN, B. N., Biochem. Biophys.
Acta, 116, 471 (1966).
(52) 3. CLELAND, W. W., Biochem. Biophys. Acta, 6’7, 104 (1963).
(Cl
)I
T‘g+KlKa
k&3
(0
(B) ((3
4. MICHAELIS,
(1913).
L., AND MENTEN, M. L., Biochem. Z., 49, 333

k3)
+yjy-
131
5. SEGAL, H. L., KACH~~AR,
16, 187 (1952).
6. BOTTS, J., AND MOR.LLES,
J. R., AXD BOYER,

J. F., Trans. Faraday

P. D., Enzymologia,

Sot., 49, 696

(1953).
which may be expressed differently by rearranging and em- 7. KING, E. L., J. Phys. Chem., 60, 1378 (1956).
ploging the relationship, k’&, = K&g 8. ARI~NS, E. J., VAN ROSSUM, J. M., AND SIMONIS, A. M.,
ArzneimitteZ-Forschung, 6, 611 (1956).
9. FRIEDEN, C., J. Biol. Chem., 239, 3522 (1964).
10. WONG, J. T.-F., AND HANES, C. S., Can. J. Biochem. Physiol.,
v x
La
40, 763 (1962).
(53) 11. MONOD. J.. WYMAN. J., .ZND CHINGEUX, J. P., J. Mol. Biol..
n‘7 12, 8s (1965). ’
12. KOSHLAND, D. E., JR., ??~METHY, G., AND FILMER, D., Bio-
1 + (B) + (c) + (B)(C) +(h + kd + kdB)(C) chemistry, 6, 365 (1966).
Kl K2 KlKs
--> k,(A) h(A)K& 13. ATKINSON, D. E., Annu. IZeo. Biochem., 36, 85 (1966).
 A Simple Method for Derivation of Rate Equations for Enzyme-catalyzed
Reactions under the Rapid Equilibrium Assumption or Combined Assumptions
of Equilibrium and Steady State
Sungman Cha
J. Biol. Chem. 1968, 243:820-825.

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