J. theor. Biol.

(1997) 187, 207–212

Closed Form Solution for Time-dependent Enzyme Kinetics

S. S*,†  C. M‡,§

*Departamento de Biologı́a Celular, Universidad Simón Bolı́var, P.O. Box 89000, Caracas
1080A, Venezuela; and ‡Centro de Fı́sica, Instituto Venezolano de Investigaciones Cientı́ficas,
P.O. Box 21827, Caracas 1020A, Venezuela

(Received on 9 September 1996, Accepted in revised form on 14 February 1997)

Based on the quasi-steady-state approximation, a closed form solution for the total time evolution of
the reactant concentrations in the basic enzyme–substrate reaction is reported for the first time. Such
a solution is given in terms of the omega function, which satisfies the transcendental equation
W(x)exp(W(x)) = x, and enables the generation of the corresponding time derivatives thus fully
characterizing the system. The agreement with results obtained from both the inner (fast region) and
outer (slow region) solutions of singular perturbation procedures is very good, but an advantage of the
present formalism is the analytic representation of the transition where the perturbation methods are
shown to be inaccurate.
7 1997 Academic Press Limited

1. Introduction Laidler (1955) who found an excess substrate

The Michaelis–Menten framework (Michaelis &
Menten, 1913) has proven to be a simple yet powerful
approach to describe enzyme processes. Its power
resides in the time-independent hyperbolic relation of
the initial velocity with initial substrate concentration
that leads to a linear double reciprocal plot (the
Lineweaver–Burk plot) from which the reaction
parameters, namely the rate constant KM and
maximum velocity vmax , can be determined. This
model is therefore fundamental to the study of
enzyme kinetics as it has made possible quantitative
estimates of enzyme characteristics, destruction and
formation rates, and the analyses of enzyme
competition and inhibition (Alberty, 1956, 1959;
Hearon et al., 1959; Segel, 1975; Fersht, 1985). It is
based on the assumption—usually referred to as the
quasi-steady-state approximation (QSSA)—that after
an initial short transient the reactant concentrations
vary slowly as if in a quasi-steady state. The actual
validity of this approximation was first discussed by
†E-mail:sschnell.taquion.ivic.ve
§E-mail: claudio.taquion.ivic.ve.
concentration to be a main requisite. Using the early
analog computers, Hommes (1962), Walter &
Morales (1964) and Walter (1966) mapped the range
of validity of the QSSA for both the irreversible and
reversible Michaelis–Menten reactions, showing
notable shortcomings for cases with large reverse
bimolecular velocity constants. Wong (1965) made an
attempt to develop a continuous description of the
transient- and steady-state phases, and concluded
that the transient state must be brief for the QSSA
to be applicable, which is obtained by increasing the
substrate/enzyme ratio. Stayton & Fromm (1979)
found the QSSA to generally hold for substrate/
enzyme ratios greater than 100 by means of
simulation modeling on a digital computer; and by
considering the time-dependent process, Schauer &
Heinrich (1979) gave a detailed analysis of the errors
resulting from the QSSA. Moreover, due to
difficulties in obtaining closed form solutions for the
system of nonlinear differential equations that
describe enzyme–substrate reactions, much effort has
been devoted to developing effective scaling and
singular perturbation techniques that lead to fairly

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208 .   . 
k1 k2
accurate solutions but make analysis extensive and S + E gh ES 04 E + P (1)
k−1
cumbersome (Bowen et al., 1963; Heineken et al.,
1967; Seshadri & Fritzch, 1980). More recently, the where S, E, ES, P denote, respectively, the free
general validity of the QSSA and the implementation enzyme, free substrate, the enzyme–substrate inter-
of perturbative methods to solve the rate equations mediate complex and product. The parameters k1 , k−1
have been extensively revised by Segel (1988), Segel & and k2 are positive rate constants for each reaction.
Slemrod (1989) and Borghans et al. (1996). These authors focused their study on reactions where
In spite of its success, the application of the the initial velocity (the rate of catalysis at which ES
Michaelis–Menten approach to estimate kinetic goes to E + P) was related to the initial substrate
constants does present some experimental problems. concentration.
For instance, it is sometimes difficult to measure By applying the law of mass action, which states
accurately the slope of a reaction curve extrapolated that reaction rates are proportional to the concen-
to zero time; furthermore, most of the information trations of the reactants, the time evolution of scheme
contained in the time evolution is wasted when only (1) can be determined from the solution of the system
initial velocities are determined. Alternative fitting of coupled nonlinear differential equations
procedures of the progress curve can significantly
d[S]
reduce the number of experimental steps; however, = −k1 [E][S] + k−1 [ES] (2)
dt
due to the lack of closed form solutions, such
attempts have made use of either the differential and d[E]
= −k1 [E][S] + (k−1 + k2 )[ES] (3)
integrated forms of the rate equations, which dt
nonetheless present the same problems of having to
estimate instant velocities by hand or use graphical d[ES]
= k1 [E][S] − (k−1 + k2 )[ES] (4)
analyses (Darvey et al., 1975). Recently, Ritchie & dt
Prvan (1996) have assessed the accuracy of the d[P]
experimental determinations of KM and vmax using = k2 [ES] (5)
dt
Monte Carlo modeling; they have concluded that the
unweighted double reciprocal plot, which has been with initial conditions at t = 0
extensively used to determine most published data, ([S], [E], [ES], [P]) = ([S0 ], [E0 ], 0, 0). (6)
can lead to unsatisfactory results thus favouring
nonlinear least-square fits to the Michaelis–Menten Since the enzyme E is a catalyst, its total
equation. concentration (free plus combined) must be a
In the present work, taking into account only the constant. This conservation law is readily obtained by
well-established assumptions of the QSSA, we have adding eqns (3) and (4):
been able for the first time to find a closed form
d[E] d[ES]
solution to describe the variations of the reactant + = 0 c [E](t) + [ES](t) = [E0 ]. (7)
dt dt
concentrations during the complete time span of the
basic enzymatic reaction, i.e. during both the fast Furthermore, at any time the sum of the concen-
initial transient and the slow quasi-steady regime that trations of the free substrate ([S]), complex ([ES]) and
follows. This solution simplifies analysis, makes it product ([P]) must be equal to the initial substrate
more consistent and gives an accurate representation concentration ([S0 ]); that is, by adding eqns (2), (4)
throughout, particularly in the region just after the and (5) it may be shown that
initial transient where most measurements are carried
out. Its accuracy depends on the validity of the QSSA, d[S] d[ES] d[P]
+ +
and it should therefore be useful in the refinement of dt dt dt
experimental methods. In Section 2, we outline the =0 c [S](t) + [ES](t) + [P](t) = [S0 ]. (8)
basic equations of enzyme kinetics and discuss the
QSSA in Section 3. The closed form solution is These two conservation laws reduce the system of
derived in Section 4 followed by a short discussion differential eqns (2)–(5) to only two equations for S
(Section 5). and ES, namely
d[S]
= −k1 ([E0 ] − [ES])[S] + k−1 [ES] (9)
2. Basic Enzyme Kinetics dt
In their seminal paper, Michaelis & Menten (1913) d[ES]
= k1 ([E0 ] − [ES])[S] − (k−1 + k2 )[ES]. (10)
proposed the basic enzymatic reaction dt
       209

It will be shown in the following section (Section 3) slow-time regimes. Furthermore, during the initial
that further simplifications can be introduced by transient, eqn (10) can then be solved to give
exploiting the QSSA.
[E0 ][S0 ]
[ES](t) =
KM + [S0 ]
3. The Quasi-Steady-State Approximation
×(1 − exp(−(KM + [S0 ])k1 t)) (t Q tC ) (17)
In its general form the QSSA states that, after an
initial fast transient, the enzymatic system enters a which, taking into consideration the form of eqn (13),
slowly changing regime where the dependent variables can be safely extended to the post-transient period.
can be assumed to be in instantaneous equilibrium. Therefore, the QSSA allows a subtle decoupling of the
This condition implies that the concentration of the differential equations describing the reaction that is
intermediate complex is in a quasi-steady state with valid throughout its complete duration. Therefore,
regard to the substrate and product, and is ensured the total solution (0 Q t Q a) can be obtained from
when the enzyme is permanently saturated with the equations
substrate; that is for
d[S] v [S]
[E0 ] = − max (18)
1. (11) dt KM + [S]
[S0 ]
[E0 ][S]
As recently recapitulated by Segel (1988) and Segel [ES](t) = (1 − exp(−(KM + [S])k1 t)) (19)
KM + [S]
& Slemrod (1989), the QSSA is based on two
assumptions. with the initial condition ([S], [ES]) = ([S0 ], 0) at
I. After the initial transient, t q tC say, the complex t = 0. These conclusions have been previously
concentration remains approximately constant due to discussed by, among others, Rubinow (1975).
enzyme saturation. Rather than finding an analytic solution for
That is, in the slow-time regime it can be taken that eqn (18), most previous theoretical work has
d[ES] concentrated on developing singular perturbation
1 0. (12) solutions in the two time regimes by means of suitable
dt
scaling methods (see, for instance, Bowen et al., 1963;
With this condition we can employ eqn (10) to solve Heineken et al., 1967; Rubinow, 1975; Seshadri &
for [ES] in terms of [S] thus Fritzch, 1980; Segel, 1988; Segel & Slemrod, 1989;
Murray, 1993; Borghans et al., 1996 and references
[E0 ][S]
[ES] = (t q tC ) (13) therein). In this context, Segel (1988) suggested two
KM + [S] time-scales: (i) the time related to the duration of the
where KM is the Michaelis–Menten rate constant initial fast transient, tC , and (ii) the time taken for a
significant change in S concentration, tS , that
k−1 + k2 characterizes the quasi-steady period. Using eqn (17),
KM = . (14)
k1 the fast-time scale can be shown to be
Substituting relation (13) in eqn (9) leads to the 1
decoupled differential equation tC = . (20)
k1 (KM + [S0 ])
d[S] v [S]
= − max (15) The slow-time scale has been determined by dividing
dt KM + [S] the total change in substrate concentration by the
where vmax = k2 [E0 ] is referred to as the maximum maximum rate of substrate change
velocity.
II. During the initial transient the substrate [S0 ]
tS = (21)
concentration hardly changes. =d[S]/dt = max
Therefore, for t Q tC , it may be assumed that
KM + [S0 ]
= . (22)
[S] 1 [S0 ]. (16) k2 [E0 ]

This assumption has the important implication that Scaling and perturbation methods to obtain sol-
eqn (15), together with the boundary condition utions in these two time-scales have been extensively
[S] = [S0 ] at t = 0, is valid in both the fast- and reviewed by Segel & Slemrod (1989).
210 .   . 

Segel (1988) has shown that the condition for [S] to reduced concentration provides a measure of the
be nearly constant during the pre-steady state period saturation of the system, since the initial velocity
implies that largely depends on it rather than on the actual
concentration, while k approximates the fraction of
tC
=D[S]/[S0 ]= 1 =d[S]/dt = max1. (23) substrate that is converted to product per unit time
[S0 ]
(Segel, 1975).
Using eqn (9) with [ES] = 0 leads to With eqns (27), (19) and the conservation laws (7)
and (8), we are in a position to elegantly reconstruct
=d[S]/dt = max = = − k1 [E0 ][S0 ]=, (24) the time evolution of all the reactant concentrations
and with the definition (20) of tC , it follows that and their derivatives during the complete duration of
the reaction (0 E t E a) as shown in Figs 1 and 2.
[E0 ]
=D[S]/[S0 ]= 1 . (25) The infinite time range has been mapped onto the
KM + [S0 ] interval (0, 1) by means of the exponential time scale
Therefore, a more general condition for the QSSA to t = 1 − 1/ln(t + e), which brings out the interesting
be valid is details of the transition between the two regimes. The
reactant concentrations have been non-dimensionally
[E0 ] scaled as follows:
1. (26)
KM + [S0 ]
[S] [E] [ES] [P]
s= , e= , c= , p= , (31)
[S0 ] [E0 ] [E0 ] [S0 ]
4. Closed Form Solution
It has been shown in Section 3 that, assuming the and their time derivatives have been normalized to
QSSA, the total time evolution of the reactants in the their maximum absolute values. The essence of the
basic enzymatic reaction is reduced to the solution of QSSA is clearly illustrated: it may be seen (curve c
eqn (18). In the present work, with the aid of a in Fig. 2) that d[EC]/dt 1 0 for t q tC , which is
powerful computer algebra system (Char et al., 1991), equivalent to enzyme saturation (curve e in Fig. 2),
we have been able to find a closed solution for this and that [S] 1 [S0 ] for t Q tC (curve s in Fig. 1); also
equation of the form the property usually measured in the laboratory as the
‘‘initial velocity’’ must be related to the slowly

0 0 11
[S0 ] −vmax t + [S0 ] changing maximum in dp/dt that occurs just after the
[S](t) = KM W exp (27)
KM KM transient (see curve p in Fig. 2).

where W is the omega function (Fritsch et al., 1973; tc ts

Corless et al., 1993). This relatively unknown 1.0
function, first introduced by Euler in 1779, satisfies
s
the transcendental equation
0.8
Scaled concentration

e
W(x)exp(W(x)) = x; (28) p

and is useful in the treatment of some nonlinear 0.6

systems, in particular those for which it is difficult to
express solutions in terms of the independent c
0.4
variables. For instance, in the present case it is well
known (Rubinow, 1975) that the integration of
eqn (18) results in 0.2

[S] + KM log([S]/[S0 ]) = [S0 ] − vmax t, (29)

but it had not been possible to derive a solution of
[S] = f(t).
Equation (27) can be conveniently expressed in the
non-dimensional form
[S '](t) = W([S'0 ]exp(−kt + [S'0 ])) (30)
where [S '] 0 [S]/KM is the reduced concentration and
k 0 vmax /KM is the first-order rate constant. The
0
0 0.2 0.4 0.6 0.8 1.0
Scaled time
F. 1. Time-dependent behaviour of the reactant concentrations
in an enzyme–substrate reaction. The infinite time range has been
mapped onto the interval (0, 1) with the aid of the exponential time
scale t = 1 − 1/ln(t + e), and the reactant concentrations have
been non-dimensionally scaled as follows: free substrate s = [S]/
[S0 ]; free enzyme e = [E]/[E0 ]; complex c = [ES]/[E0 ] and product
p = [P]/[S0 ]. The fast, tC , and slow, tS , time-scales are also shown.
       211

tc ts in the analytic description of the transition that takes

1.0
p place in the interval tC Q t Q tS where perturbation
solutions can be seen to exhibit a significant
discrepancy, particularly near the maximum. An
0.5
accurate representation of this interval is important
Scaled derivative

inasmuch as most measurements in the laboratory are

c performed within its duration.
0
0 0.2 0.4 0.6
e
Scaled time
–0.5
s time evolution of an enzyme–substrate reaction,
–1.0
F. 2. Evolution of the time derivatives of the reactant
concentrations in an enzyme–substrate reaction. The infinite time
range has been mapped onto the interval (0, 1) with the aid of the
exponential time-scale t = 1 − 1/ln(t + e), and the derivatives have
been normalized to their maximum absolute value. s = d[S]/dt;
e = d[E]/dt; c = d[ES]/dt and p = d[P]/dt. The fast, tC , and slow,
tS , time-scales are also shown.
In Fig. 3 we compare the closed form solution for
the complex concentration with results obtained in
the fast (inner solution) and slow (outer solution)
scale regions by the singular perturbation procedures
described by Segel & Slemrod (1989). The agreement
of the closed form solution with the perturbation
inner solution for t Q tC and the outer solution for
t q tC to order [E0 ]/[S0 ] is, as expected, very good.
However, an advantage of the closed form is apparent
0.5
o i
0.4
Scaled concentration
0.8 1.0
5. Discussion
The main contribution of the present work is the
discovery of a closed form solution to describe the
which, consistent with the QSSA, is not the ‘‘exact’’
solution but should be accurate for [E0 ]/
(KM + [S0 ])1. We also take the opportunity to
emphasize that subject to this constraint, which
applies in many of such reactions in biochemistry, the
QSSA allows the derivation of a solution that is valid
in both the initial transient and the subsequent
quasi-steady state. Moreover, the time derivatives
of the reactant concentrations are directly obtained
from the proposed closed form solutions leading to
a complete characterization of the system, which is
invaluable in its analysis and depiction. Therefore,
the usability of the Michaelis–Menten model is
reinforced in the context of uncompetitive enzymatic
reactions. We plan to extend the present approach in
the near future to reactions with competitive
substrates.
In the light of the conclusions of the work by
Ritchie & Prvan (1996), the present formalism also
brings forth new perspectives in the implementation
of experimental techniques to determine kinetic
parameters. Traditional methods involve the
measurement of instantaneous initial velocities as a

function of the initial substrate concentration, which

c in practice is usually reduced to estimates of
0.3 ‘‘average’’ initial velocities shortly after the initial
transient. As discussed by Darvey et al. (1975), there
0.2
can be difficulties to estimate these quantities from
measurements. A new experimental approach could
be envisaged where, for a specific initial substrate
0.1 concentration, its decay is measured as a function of
time and then fitted with the present closed form
tc ts
solution to obtain the maximum velocity, vmax , and the
0
0 0.2 0.4 0.6 0.8 1.0 Michaelis–Menten rate constant, KM . Moreover, the
Scaled time
traditional methods only estimate these two par-
ameters from which it is not possible to solve the three
F. 3. Comparison of the present closed-form solution for the
complex concentration (curve c) with those obtained by singular rate constants (k+1 , k−1 and k+2 ) that fully
perturbation methods to order [E0 ]/[S0 ]. It may be appreciated that characterize the reaction. In the proposed method-
the agreement with the inner solution (curve i) during the fast ology, a complementary fitting of the temporal
transient (t Q tC ) and with the outer solution (curve o) for t q tS
is very good. However, data from the perturbation methods are variation of the free enzyme (or complex) concen-
noticeably different in the important transition interval tC Q t Q tS . tration would help to solve this problem.
212 .   . 
Part of this work was carried out during a visit of S.
Schnell to the Centre for Mathematical Biology, Math-
ematical Institute, University of Oxford, in August 1996.
The hospitality of Dr P. K. Maini and his revision of the
manuscript are both gratefully acknowledged.
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