A GUIDE TO. . .

A guide to the Michaelis–Menten equation: steady state

and beyond
Bharath Srinivasan
Mechanistic Biology and Profiling, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK

Keywords The modern definition of enzymology is synonymous with the Michaelis–

asymptotes; enzyme kinetics; initial velocity;
Michaelis–Menten; quasi-steady state; rapid
equilibrium; rectangular hyperbola; steady
state
Correspondence
B. Srinivasan, Mechanistic Biology and
Profiling, Discovery Sciences, R&D,
AstraZeneca, The Darwin Building, 310
Milton Road, Cambridge CB4 0WG, UK
Tel: +351 920122242
E-mail: bharath.srinivasan@astrazeneca.com
(Received 12 May 2021, revised 1 July
2021, accepted 15 July 2021)
doi:10.1111/febs.16124
Menten equation instituted by Leonor Michaelis and Maud Menten. Most
textbooks, or chapters within, discussing enzymology start with the deriva-
tion of the equation under the assumption of rapid equilibrium (as done
by Michaelis–Menten) or steady state (as modified by Briggs and Haldane)
conditions to highlight the importance of this equation as the bedrock on
which interpretation of enzyme kinetic results is dependent. However, few
textbooks or monographs take the effort of placing the equation within its
right historical context and discuss the assumptions that have gone into its
institution. This guide will dwell on these in substantial detail. Further, this
guide will attempt to instil a sense of appreciation for the mathematical
curve rectangular hyperbola, its unique attributes and how ubiquitous the
curve is in biological systems. To conclude, this guide will discuss the limi-
tations of the equation, and the method it embodies, and trace the journey
of how investigators are attempting to move beyond the steady-state
approach and the Michaelis–Menten equation into full progress curve,
pre–steady state and single-turnover kinetic analysis to obtain greater
insights into enzyme kinetics and catalysis.

Introduction scientific community for more than a century. The

“Essentially, all models are wrong, but some are use-
ful”
G.E.P. Box and N.R. Draper in Empirical Model-
Building and Response Surfaces (Wiley, New York
(1987))
Michaelis and Menten equation (MM equation) has
dominated biochemistry for more than a century after
its seminal introduction in a paper published in 1913
in the journal Biochemische Zeitschrift, a predecessor
of FEBS Journal [1]. Hence, publishing this guide in
FEBS Journal would represent an apt dedication to
the unmatched service rendered by this journal to the
paper was initially published in German with the title
‘Die Kinetik der Invertinwirkung’ [2] and has now
been translated into English [3,4]. The equation has
almost assumed a sacrosanct status in its ability to
predict and sustain biochemical research and is an
example of how diligent science can stand the scrutiny
of time and technological advancement. The equa-
tion was an attempt to explain the behaviour of an
enzyme (binding of substrate and subsequent catalysis)
long before the exact nature of enzymes as proteins
and catalysis happening at the active site was an
accepted fact. Moreover, the work and its conclusions,
which were based upon studies carried out on a single
enzyme (invertase), was subsequently shown to be

Abbreviations
FLA, free ligand approximation; KIE, kinetic isotope effect; MM, Michaelis–Menten; QSS, quasi-steady state; RSA, reactant stationary
assumption; SS, steady state.

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B. Srinivasan Enzyme kinetics and the Michaelis–Menten equation

applicable to most of the enzymes. Little did Leonor would have to be appreciated that some of the reac-
Michaelis, a scientist working as an unpaid professor, tions are so slow in the absence of catalysts that their
and Maud Menten realize that their equation would rates can never be estimated even with increased tem-
become synonymous with the biochemistry of enzyme peratures and high pressures [7]. To study and under-
action. This guide attempts to trace the origin of the stand such diverse timescales, a variety of techniques
equation, its place within the broader field of kinetic would have to be employed. This manuscript is inter-
timescales, dwells into its mathematical elegance and ested in those reactions that happen on timescales of a
discusses the immense body of work whose culmina- little more than one turnover per hour to a few 1000
tion was this cogent mathematical expression per second. A whole spectrum of techniques can moni-
(Table 1). Building upon that, this guide explores the tor reactions that happen in the span of hours, min-
journey beyond the steady-state assumption and briefly utes and/or seconds. Examples include the
looks at the rich information content in the pre–steady conventional UV-Vis spectrophotometers, fluorescence
state, single turnover and full progress curve modelling spectrophotometers, mass-spectrometry and analytical
approaches in enzyme kinetics. fractionation methods, to name just a few. However,
reactions happening on the timescale of say 1 to sev-
eral 1000s per second (second to millisecond time-
Kinetics, timescales of chemical
scales) require techniques for rapid mixing and
reactions and the steady state
quenching. For this, continuous-flow and stopped-flow
Biochemical reactions happen over a wide range of mixing devices have been used with dead times as low
timescales. The width of the scale can be appreciated as 30 μs. Below this limit, the viscosity of the solution
by perusing through the work of Richard Wolfenden, plays a major role in determination of proper mixing
where he shows how biochemical reactions can span of substrate and enzyme solutions. For reactions that
from 4 billion years (10−18 s−1) for arginine decarboxy- happen on the timescale of microseconds to hundreds
lation in the absence of catalyst to ~ 103 s−1 (approxi- of milliseconds, relaxation techniques are the preferred
mately 1021 fold enhancement for kcat/knon!) [5,6]. It approach. The latter is a method for perturbing the
equilibrium or steady state by a sudden change in an
external condition such as temperature or pressure.
Table 1. Summary timeline of the various discoveries and
The relaxation of the system back to equilibrium
improvements of the initial MM treatment.
reveals useful information about the kinetics of the
Researcher/s Year Postulate reaction. These latter methods were preferred because
C. O’Sullivan and 1890 Studies establishing enzyme kinetics
of the ease of modelling such systems by linearization
F. Tompson as a distinct discipline different from of differential equations near equilibrium.
A.J. Brown 1892 chemical kinetics
H.E. Fischer 1894
G. Tammann 1895
Simplicity and elegance of a
E. Duclax 1899 mathematical curve: the rectangular
A.J. Brown 1902 Enzyme saturation hyperbola
V. Henri 1903 Invertase kinetics: initial velocity
S.P.L. Sǿrenson 1909 pH scale ([H+] ions)
Even before dwelling into the MM equation, this sec-
A.V. Hill 1910 Haemoglobin cooperativity tion attempts to provide an aesthetic appreciation and
L. Michaelis and 1911 Effect of [H+] ions on enzyme activity expound on the unique attributes of the mathematical
H. Davidsohn curve that the equation represents.
L. Michaelis and 1913 Invertase kinetics: Rapid-equilibrium Curves are beautiful. Any student of mathematics
M. Menten assumption, effect of pH, who has had the opportunity to interact with functions
mutarotation and affinity of substrate
such as an epitrochoid, limaçon, lemniscate and car-
for enzyme (Ks)
D.D. van Slyke 1914 Irreversibility of ES complex formation
dioid would know this (Fig. 1A). Curves not only
and G.E. Cullen and application of qSSA for urease embody aesthetic beauty that appeals to the visual
reaction. sense but are also conceptually beautiful, providing a
G.E. Briggs and 1925 Steady-state assumption, affinity of relationship between an independent variable and a
J.B.S. Haldane substrate for enzyme (Km) dependent one to provide a cogent and pithy descrip-
H. Lineweaver 1934 Double reciprocal linear transformation tion of a complex phenomenon. One family of curves,
and D. Burk for parameter estimation.
which have fascinated humankind for a long while
B. Chance 1943 Trapping the ES intermediate in
peroxidase by stopped flow
now, are conic sections. A cone can be sectioned in
several different ways to give rise to three unique conic

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Enzyme kinetics and the Michaelis–Menten equation B. Srinivasan

Fig. 1. (A) Beauty of mathematical curves shown using two representative curves, cardioid and lemniscate (B) Conic sections. The angle of
the plane intersecting the cone will determine the different conic sections as shown on the right using ‘textbook’ representation. (C) A plot
of hyperbola on a 2-dimensional Cartesian coordinate system. (D) The rectangular hyperbola exemplified by the Michaelis–Menten
equation showing the first-order zone and the zero-order zone, respectively.

sections depending on the angle of the plane that inter-
sects it (Fig. 1B): an ellipse (a circle is a special case
thereof), a parabola and a hyperbola. The mathematics
of ellipse and parabola has been discussed extensively
elsewhere. Here, we focus on gaining an appreciation
for the beauty of the hyperbola. Hyperbola, in its sim-
plest form and in a Cartesian coordinate system, can
be expressed as (Fig. 1C).
1
x
y ¼ 1ory ¼ :
x
Notice that the multiplicative total of the two vari-
ables is a constant and hence the two variables are
inversely related to one another. Stated differently, an
increase in the magnitude of one of the variables will be
compensated for by a decrease in another variable.
Hence, this curve is also called saturating hyperbola,
asymptotic regression, or a decelerating curve (Fig. 1B).
Another property of this curve is that independent vari-
able x can never be zero since division by zero is unde-
fined. The curve is always governed by two parameters
and can never approach the asymptotes (Fig. 1C). A
hyperbola can have two measurable attribute: the
asymptotes, and the rate at which the dependent param-
(1) eter approaches the asymptote as a function of increas-
ing independent parameter. The curve is called a
rectangular hyperbola when the asymptotes are perpen-
dicular (intersect rectangularly). A rectangular hyper-
bola is a curve that describes a plethora of biological
processes including dependence of reaction velocity on

6088 The FEBS Journal 289 (2022) 6086–6098 ª 2021 Federation of European Biochemical Societies.

B. Srinivasan
substrate concentration in biochemistry, % maximum
effect of drug concentration in pharmacology, photo-
synthetic rate/growth rate on light intensity/nutrient
level in physiology, microbial growth rates in aqueous
environments to the concentration of a limiting nutrient
(Monod equation), type II functional response as
emphasized by Holling’s disc equation showing the rela-
tionship between prey density and prey consumption by
predator, several instances in population ecology, Lang-
muir isotherms and many more [8].
The expression for a rectangular hyperbola (Eqn 2)
takes into account both the maximum height of the
asymptote and the rate of approach towards it and is
written as follows
a
x
y¼ , (2)
ðb þ xÞ
where y is the dependent variable, x is the independent
variable, a is the asymptote and b is the parameter
defining the rate of approach to asymptote [8].
Depending on the biological phenomenon that is
defined by this equation, the dependent variable, inde-
pendent variable and the rate of approach parameter
can assume different nomenclature and can be com-
posite functions.
The equation defined by Leonor Michaelis and
Maud Menten (and before that by Victor Henri) is a
right rectangular hyperbola that has limits of Vmax
and −Km (Eqn 4) (Fig. 1D). Stated another way, the
relationship between velocity and substrate concentra-
tion variation is a constant exemplifying the beauty of
the equation. The curvature of the function that is
defined by this equation remains invariable regardless
of the absolute values of Km and Vmax. Hence, the
ratio between substrate concentrations for two velocity
fractions will remain constant for any enzyme that fol-
lows the Michaelis–Menten kinetics. For example, the
ratio of substrate required for 90% of Vmax to the sub-
strate required for 10% of Vmax is always 81 irrespec-
tive of the actual identity of the enzyme, provided that
the enzyme follows Michaelis–Menten kinetics [9].
However, empirical sciences are rarely as accurate as
theoretical constructs, and hence, a value of 15% for
the ratio would still be considered as representing the
rectangular hyperbola. This can be used as a diagnos-
tic trait for the experimental data obtained. Any sub-
stantial deviation from 81 would be indicative of non-
MM kinetics showing either positive or negative coop-
erativity. It has to be highlighted that the parameter
Vmax represents the dependent variable that
approaches the asymptote and the parameter Km rep-
resents the rate of approach to the asymptote in the
context of the hyperbola.
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Enzyme kinetics and the Michaelis–Menten equation
Another thing that emerges from the MM equation,
and the resultant plot, is the zonation and the meaning
of such zones. At very low substrate concentrations,
the velocity is linearly proportional to the substrate
concentration. Stated otherwise, velocity is first order
vis-à-vis substrate concentration. This is called as the
first-order zone and enables the most accurate determi-
nation of the first fundamental steady-state kinetic rate
constant kcat/Km. At very high substrate concentra-
tions, the rate of change of velocity as a function of
substrate concentration variation is negligible
approaching asymptomatically a constant called kcat.
kcat is the second fundamental steady-state kinetic rate
constant, and this zone is called the zero-order zone.
The zone between the zero order and the first order is
variously called as mixed-order or pseudo-first-order
zone and is often referred to as the zone for the proper
estimation of Km. However, it would have to be
emphasized that Km is merely a ratio of the parameters
estimated at the zero-order zone and the first-order
zone.
What is the MM equation and why was
it derived
Michaelis and Menten used the reaction of invertase
(see Looking back: Historical context of this equa-
tion’s derivation) and the scheme specified below to
arrive at the equation.
Scheme 1
The equation, as it appears in the paper by Michae-
lis and Menten [10], is as follows
½S
v¼Cϕ , (3)
½S þ k
where v is initial velocity, C is kcat multiplied by a fac-
tor (conversion factor computing substrate to product
conversion from optical rotation), ϕ is the total
enzyme concentration, [S] is substrate concentration
and k is Ks (the equilibrium dissociation constant of
substrate with the enzyme).
The modern variant of this equation is shown as
ðVmax
½SÞ
v¼ , (4)
ðKm þ ½SÞ
where v is velocity, Vmax is maximum velocity when all
the enzyme is complexed to the substrate, [S] is sub-
strate concentration and Km is the pseudo-equilibrium
constant called Michaelis–Menten constant.
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Enzyme kinetics and the Michaelis–Menten equation
Why did Michaelis and Menten attempt to derive
this equation? Though the answer to this question is
straightforward, I have noticed that several undergrad-
uates (and even graduate students) are confused when
posed this question. It suddenly dawns upon them that
they had been trying to understand ‘how’ the equa-
tion was derived before understanding ‘why’ the
attempt was made in the first place. The answer to this
conundrum is simple. The equation is an attempt at
expressing the unmeasurable parameters in terms of
the measurables. As shown in the Scheme 1, enzyme
forms a complex with the total substrate to give rise to
the enzyme–substrate complex. Three distinct rate con-
stants define this model: (a) The second-order associa-
tion rate constant k1 for the formation of the enzyme–
substrate (ES) complex from free enzyme and total
substrate (formation of this complex partitions the
total substrate into free substrate and bound substrate;
however, the problems arising because of this are
avoided by using an excess of the substrate in order
for the total substrate concentration to remain invari-
able in spite of complexation). (b) The first-order dis-
sociation rate constant k−1 for the dissociation of ES
back to E and S; and (c) the first-order catalytic rate
constant k2 for the breakdown of ES to E + P. In the
above scheme, the initial rate v0 of the reaction is
dependent on the concentration of ES.
v0 ¼ k2 ½ES:
At this point, it would have to be emphasized here
that the purpose of the equation is to quantify the
velocity (a vectorial quantity with attributes of magni-
tude and direction) rather than speed (a scalar quan-
tity defined by magnitude alone). The velocity of the
reaction can be estimated by the rate of change of ES
in the direction of product formation (processes that
convert ES complex to product normalized by those
that break it down back into substrate). However, the
intermediate ES complex cannot be observed and can
only be assessed indirectly as the rate of product for-
mation or substrate dissipation. The equation was
derived as an attempt at expressing the unmeasurable
ES complex in terms of measurable parameters such as
total substrate concentration, total enzyme concentra-
tion and the entire algebraic exercise was undertaken
to enable this.
During the times of Michaelis and Menten, nonlinear
least-square fits were not feasible. Hence, they did not
estimate the kinetic parameters by plotting v versus [S]
to avoid the difficulty of estimating the location of the
asymptote v = Vmax. Rather, they plotted the data as v
versus log [S]/Km based on their knowledge of effect of
pH on the dissociation of a weak acid. Parameters
6090 The FEBS Journal 289 (2022) 6086–6098 ª 2021 Federation of European Biochemical Societies.
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B. Srinivasan
estimated thus have stood the test of time as shown in a
recent paper where modern techniques of data analysis
have been used [3]. However, current methodologies for
parameter estimation rely on least-square nonlinear fits
on the v versus [S] plots or full time-course simulations
of the progress curves [11–16].
Assumptions and approximations:
obtaining the equation under initial
velocity conditions
Biological systems are complex, dynamic, nonlinear
and, often, unpredictable. A lot is unknown about the
physical laws that govern their behaviour. In such situ-
ations, it is difficult, if not impossible, to write down
detailed equations that can accurately describe the sys-
tem under study. Moreover, precise analytical treat-
ment yielding an exact solution could be expensive
and, oftentimes, impossible. In such situations, an
approximate solution using trial-and-error–based
numerical approaches is attempted. These solutions
may not be accurate. However, under reasonable
boundary conditions and with approximations and
assumptions that are physically realistic, we might
obtain solutions that do not lead to type I errors. Sta-
ted otherwise, a numerical approach to solving a com-
plex equation results in an approximation of the
biological system under study within acceptable error.
As we will see below, the MM equation represents a
special case of nonlinear dynamical system where
assumptions and approximations were judiciously
applied to obtain realistic solutions under conditions
of initial velocity and rapid equilibrium during the
time of Michaelis and Menten.
The first assumption under which the MM equa-
tion is valid is called initial velocity (Table 2). This
means the velocity of an enzyme reaction at its incep-
tion (time zero). Theoretically, this instantaneous rate
can be estimated by drawing a tangent to the progress
curve at t = 0 and estimating the slope of the tangent.
Why did Michaelis and Menten take recourse to initial
velocity as an assumption? Most investigators working
on trying to understand the kinetics of invertase took
recourse to modelling the complete time course of the
reaction. However, this led to complexities such as
nonlinear progress curves and the later realization that
the nonlinearity is due to a phenomenon called pro-
duct inhibition (the first biochemical instance at
attempts to characterize inhibition!). This realization
entailed the modelling of this phenomenon, complicat-
ing the description of the algebraic rate equations. To
avoid this, Michaelis and Menten (and Henri before
them) rightly argued that if the reaction velocity was
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B. Srinivasan Enzyme kinetics and the Michaelis–Menten equation

Table 2. Summary of the assumptions and the range of conditions that need to be maintained to obtain reliable results when using MM
equation.

Assumption/Conditions Boundary conditions Implications

Initial velocity d [P]/dt = 0 at t = 0

Enzyme concentration
Substrate concentration
Reactant stationary assumption/
Free ligand approximation
Quasi-steady-state assumption
Rapid-equilibrium assumption
Rectangular Hyperbola
a
Theoretical.
b
Practical.
2 2 a
No product inhibition, no enzyme inactivation, linear progress curves. Initial
[P] < 5%[S]totalb velocity, in most cases, span the initial transient and quasi-steady state
E ≪ S The enzyme should be multiple orders of magnitude lesser than substrate
S≫E The substrate should be multiple orders of magnitude greater than enzyme
concentration to maintain RSA/FLA
[S]total = [S]free In the initial transient, when the ES complex concentration builds up to steady
state and the free enzyme concentration decreases, the total substrate can be
still approximated to free substrate because of the excess of substrate vis-à-
vis enzyme
d[ES]/dt = 0; This assumption simplifies the algebraic derivation of rate equation (d[P]/
Km = (k2 + k−1)/k1 for dt = (kcat[S])/(Km + [S])) and expresses it in terms of the known parameters.
Scheme 1 This assumption was used for enzyme kinetics by G.E. Briggs and J.B.S.
Haldane
E and S are in rapid This assumption was instituted by Michaelis–Menten and is true for cases
equilibrium with ES; where the catalysis is rate limiting. That is, enzyme and substrate equilibrate
Ks = k−1/k1 for with the enzyme–substrate complex faster than the rate at which the complex
Scheme 1 breaks down to products
[S]0.9/[S]0.1 = 81 The substrate versus velocity plot should be a rectangular hyperbola. That is,
the ratio of substrate concentrations for any two fractions of maximal velocity
should be a constant. Any deviation from a hyperbola should be appropriately
treated

to be measured at t = 0, there would be no product
formed thus avoiding the complication arising because
of product inhibition. In retrospect, we also know that
studies carried out under initial velocity conditions
avoid problems associated with substrate depletion
and enzyme inactivation. Hence, it was this realization
(of initial velocity) that spurred a century of fruitful
enzyme kinetic investigations.
However, as an experimentalist, it would have
immediately dawned upon you that measuring velocity
at time t = 0 is an impossible exercise to undertake
because d[P]/dt = 0 at t = 0. For all practical pur-
poses, and taking into consideration the law of mass
action and the respective affinities of products and
reactants, initial velocity can be redefined as velocity
monitored under conditions where ‘substantial’ deple-
tion of substrate or formation of products has not
taken place. The ‘substantial’ zone has been histori-
cally assigned as < 5% of the initial substrate concen-
tration. However, several investigators use percentages
that vary from 5% to as high as 20% in some cases as
their definition of initial velocity! Though, there is no
hard and fast rule and one can get away with these
high conversion percentages if the affinity of the pro-
duct for the enzyme is not high, being conservative will
always ensure results that are compliant with the
model Michaelis and Menten proposed.
The second assumption suggests that the model is
applicable only under the conditions of rapid equilib-
rium (Table 2). Under these conditions, the enzyme–
substrate complex is in equilibrium with free enzyme
and free substrate, respectively. This assumption essen-
tially states that the catalytic step (ES → E + P) is rate
limiting in nature. In the absence of catalysis happen-
ing on the timescale of ES equilibration, ES and E + S
would reach an equilibrium, with an equilibrium asso-
ciation constant of Ka = k1/k−1. The reciprocal of this
is the equilibrium dissociation constant Ks (Ks = 1/
Ka = k−1/k1). Michaelis and Menten were the first to
explain that the term Ks in their equation is an indica-
tion of the apparent affinity of the enzyme for the sub-
strate. As noted above, the validity of this equation is
dependent on the substrate reaching equilibrium with
enzyme to form the enzyme–substrate complex on a
timescale much faster than the formation of product.
Almost simultaneously with the work done by
Michaelis and Menten, Donald Van Slyke and Glenn
E. Cullen [17], working at the Rockefeller Institute,
gave us the concept of an irreversible [ES] complex
where kcat ≫ koff. This ensures that the [ES] complex,
once formed, will go all the way to form [EP] and does
not dissociate back into free enzyme and substrate.
The irreversibility of the [ES] complex formation is a
physical impossibility because most of the enzyme–

The FEBS Journal 289 (2022) 6086–6098 ª 2021 Federation of European Biochemical Societies. 6091

Enzyme kinetics and the Michaelis–Menten equation
substrate binding is mediated by noncovalent interac-
tions dominated by hydrogen bonding and electrostatic
effects. However, by speculating along these lines
(likely influenced by Van Slyke’s training in Fischer’s
laboratory and influenced by the lock and key model
that the latter proposed), they opened the possibility
of experimenting with quasi-steady-state assumption
later taken up by George Edward Briggs and John
Burdon Sanderson Haldane [18].
So what is quasi-steady-state assumption (also called
pseudo-steady state)? Steady-state assumption (SSA),
as the name suggests, is the state where the concentra-
tion of some molecular species remains invariable.
Quasi-steady-state assumption (QSSA), as a minor
modification of the SSA, describes the kinetics of sys-
tem where, after an initial fast transient or burst, the
concentration of an intermediate molecular species is
invariant (Table 2). In this case, the invariant species is
the enzyme–substrate complex. This is a case of time-
scale separation which, by eliminating the fast compo-
nents of a reaction, results in simplification of the
system’s description [19,20]. The condition for QSSA is
that ET ≪ (ST + Km) (where ET is total enzyme, ST is
total substrate and Km is the pseudo-equilibrium con-
stant of the substrate for the enzyme as discussed
below). The QSSA is also called as standard quasi-
steady-state assumption (sQSSA) to differentiate it
from total quasi-steady-state assumption (tQSSA). The
latter is a special modification of the sQSSA that takes
into account both ET ≪ (ST + Km) and ST ≪ (ET +
Km) [21–23]. The tQSSA enables the modelling of
in cellulo biochemical system with greater accuracy.
The sQSSA treatment invariably leads to an equilib-
rium dissociation constant Km that is defined as
k2 + k−1/k1 for Scheme 1. Hence, as a cursory perusal
of conventional biochemistry textbooks would confirm,
the expression for the initial velocity remains the same
(Eqn 4) across the rapid equilibrium versus steady-
state assumption with minor difference in the way the
equilibrium dissociation constant is defined [Ks (koff/
kon) and Km (kcat + koff/kon), respectively]. However, it
would have to be appreciated that the equilibrium dis-
sociation constant’s expression can change dramati-
cally for schemes other than Scheme 1. In fact,
Scheme 1 represents a physically unrealistic model
because the chemical transformation step and the sub-
sequent product dissociation steps are treated as a
composite occurrence bundled and represented by a
single rate constant k2. This, obviously, is not true and
is merely a tool to simplify the algebra. Moreover,
occurrence of numerous distinct intermediates, during
the transformation of substrate to product, is com-
pletely ignored. To reiterate, for the steady-state
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B. Srinivasan
assumption to be valid, the enzyme concentration
should be much less than the substrate concentration
or Km of the enzyme for substrate or both.
½E0 =ð½S0  þ Km Þ ≪1:
In conclusion, the term Km, also known as the
Michaelis constant, is a pseudo-equilibrium constant
and extreme care should be exercised in interpreting its
meaning. Of late, there is consensus in literature that
treatment of Km as an equilibrium dissociation con-
stant is too naı̈ve and can lead to substantial errors if
employed to model networks in systems biology. For
Scheme 1, Km must be equal to or greater than KD
[24]. However, for any minor variations in the scheme,
the relationship between the different micro rate con-
stants become rather complex and Km can be equal to,
greater than or less than KD [25].
Another assumption that has not been appreciated
much over the years is the reactant stationary assump-
tion (RSA) (also known and free ligand approxima-
tion, FLA) [26] (Table 2). This assumption states that
the total ligand concentration remains invariable in
spite of complexation of some fraction of the substrate
pool by the enzyme. Stated differently, [S]free = [S]total
as the ES complex reaches steady state during the ini-
tial transient. This is so because of the large excess of
substrate concentration. However, this assumption has
the potential to fail in cases where the Km of the sub-
strate for the enzyme is equivalent or less than the
quantity of the enzyme used in the assay. To address
this, investigators have attempted to derive the equa-
tion with no implicit assumption that the total sub-
strate concentration is equal to the free substrate
concentration. This equation is quadratic in nature
and shifts from a hyperbola to a rectangle with
increasing affinity of the substrate for the enzyme at a
fixed enzyme concentration [27,28]. Evolution, by sys-
tematic tweaking of residues at the active site, has
taught us that a substrate with high affinity for its cog-
nate enzyme is contraproductive in catalytic terms and
hence, hardly, if any, substrates display this behaviour.
Having said that, this has been a persistent field of
investigation in inhibitor discovery where, at later
stages in the drug-discovery process, inhibitors start
showing affinities that are equal to or less than the
total enzyme used in an assay mix [28].
Strict adherence to the law of mass action is another
unique attribute that defines the derivation of the MM
equation. Victor Henri was a strong advocate of this
and wanted his modelling of the reaction kinetics of
invertase to adhere to the laws of physical chemistry.
Though this is clearly the case when modelling the

B. Srinivasan
kinetics of the enzyme activity carried out within a test
tube, translation of this law to intracellular enzymes
becomes tricky. The law of mass action requires the
enzyme and the substrate to diffuse freely to each
other for the purpose of complex formation. Of late,
studies carried out on the nature of cytoplasm within
a cell indicates that aspects of macromolecular crowd-
ing and phase separation make the interior distinctly
viscous (with gel-like properties) obstructing free diffu-
sion of metabolites and proteins as a regulatory
modality [29,30]. Further, the total concentration of
the enzyme also undergoes constant change in the cell
[31]. This observation demonstrates that the MM
equation likely is not applicable when modelling intra-
cellular enzyme kinetics and appropriate modifications
might be required to use it in latter cases [32,33].
Another assumption that would have to be rigor-
ously scrutinized is the irreversibility of the product
formation step. In most enzymology textbooks, the ES
to P transformation is indicated as being irreversible
(and for good reasons). This simplification is necessary
for obtaining an analytic solution. However, it would
be worth considering that this might not be true in all
case scenarios. In general, the assumption of irre-
versibility can safely be considered in situations where
the concentration of substrate is in far excess of pro-
duct (and if the affinity of the substrate is better for
the enzyme than that of the product), if the energy
released in the reaction is very large (ΔG ≪ 0) and if
the product formed is prevented from being accumu-
lated under initial velocity conditions.
A major aspect to be aware of when employing the
MM equation is the realization that it is a determinis-
tic model and applies to cases when the number of
molecules constituting the system is large and is
assumed to be homogenous. This can approximate the
in vitro biochemical reaction set-up using the ordinary
differential equation embodied by the MM equation.
On the contrary, most in vivo biochemical systems are
often constituted by a very small number of chemical
species where stochastic effects will have a major role
to play in modelling the system using discrete stochas-
tic modelling. The latter requires the specification of
volume to compute the number of particles instead of
using concentrations. Stated slightly differently, the
rate constants kcat, kassociation and kdissociation represent
population estimates and can deviate considerably
when measured for either individual or a small number
of molecules. Further, in in vivo biochemical systems,
the rate constants themselves can change either tempo-
rally or spatially depending on concentration of the
reacting species across time or compartments. Further,
care must be exercised when interpreting the outcome
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Enzyme kinetics and the Michaelis–Menten equation
from MM equation of a reaction that is part of a lar-
ger network of reactions [34].
Having looked at the assumptions/approximations,
one should not forget that the institution of this equa-
tion ushered in an era of steady-state kinetic analysis
where several different enzymes were interrogated
under initial velocity conditions and the field of enzy-
mology flourished. The parameters kcat and Km facili-
tated ease of communication among biochemists and
enzymologists as a comparative scale to rank-order
enzymes in terms of their catalytic efficiency (kcat/Km),
rate enhancement (kcat/knon) and catalytic proficiency
((kcat/Km)/knon) (the latter two only in case an estimate
of knon is known, assuming the mechanism within an
enzyme’s active site and in solution in the absence of
enzyme is same) [6]. In the next section, we take a
sneak peek at the historical context of this equation’s
derivation.
Looking back: Historical context of
this equation’s derivation
Invertase (EC 3.2.1.26) catalyses the hydrolysis of the
disaccharide sucrose into hexoses glucose and fructose
(Scheme 2). This enzyme and the studies carried out on
it form the backbone of quantitative enzyme kinetics.
Invertase was chosen for kinetic studies because of
its ease of purification (yeast secretes this enzyme into
the extracellular medium) and the convenience of mea-
suring the catalysed substrate to product transition
using a polarimeter [substrate sucrose and the products
glucose and fructose rotate the plane of polarized light
in opposite directions (inversion)].
Before the efforts culminated finally as the MM
equation, a long and illustrious line of investigators
contributed to quantitatively understanding the prob-
lem of the invertase catalysed reaction [35] (Table 1).
Those initial attempts were focussed on understanding
and modelling the full progress curves of the substrate
to product conversion by the enzyme in the absence of
well-crystallized thinking on aspects such as enzyme
inactivation and product inhibition, to name but a
few. This was reflected constantly in the works of the
various investigators. Cornelius O’Sullivan and Freder-
ick Tompson (1890) [36], Adrian J. Brown (1892), G.
Tammann (1895) and Emile Duclax (1899) debated
extensively on the nature of enzyme kinetics vis-à-vis
chemical catalysis (mainly acid hydrolysis) and the
relationship of the velocity on substrate concentration
[37] (Table 1).
6093

Enzyme kinetics and the Michaelis–Menten equation
In 1902, Adrian J. Brown proposed, for the first time,
that the saturation kinetics of invertase can be explained
by enzyme–substrate complex formation that cannot
increase as a function of increasing substrate beyond a
threshold substrate concentration [38,39]. This was a
very critical insight that would define the bifurcation
point between chemical kinetics and enzyme kinetics
and which would have important role to play in the
derivation of the Michaelis–Menten equation subse-
quently. Further, this hypothesis also resembled the
lock and key model (Schlüssel-Schloss-Prinzip) put
forth by the German Chemist Hermann Emil Fischer in
1894 [40]. As will be discussed later, Emil Fischer had
an immense influence on both Victor Henri and
Michaelis’s work on enzyme–substrate interaction.
However, the major criticism of Brown’s work was its
incompatibility with principles of physical chemistry
and its lack of explicit treatment of product inhibition.
It was Victor Henri who devised the first set of defini-
tive equations that formed the basis for the subsequent
MM equation. His deft combination of empirical and
theoretical approaches from the toolkit of biochemistry
and mathematics laid the foundation of this equation on
a firm footing. The influence was so much so that a few
prominent voices in the enzymological community have
repeatedly insisted on calling the MM equation as
Henri–Michaelis–Menten equation. Henri carried out
diligent experiments with invertase first followed by
elastase, emulsin and amylase, and went ahead with
modelling the reaction rate in strict conformity with
laws of physical chemistry and principles of mass action.
Henri, in agreement with the practise prevalent then,
tried modelling the kinetics as a function of time and
came up with the following equation
dx=dt ¼ ½K3 ða  xÞ=½1 þ mða  xÞ þ nx, (5)
where x is the concentration of accumulated product,
dx/dt is the rate of change of product formation as a
function of time, K3 is a composite constant that is a
product of Kmϕ (K is the rate constant, m is the equi-
librium constant and ϕ is the rate in terms of enzyme
concentration), a is initial sucrose concentration and n
is equilibrium binding constant for the product. By inte-
gration, he obtained an expression for the progress of a
reaction. Henry was also aware of the simplification that
monitoring reaction rates at time = 0 would bring to
this equation but did not pursue that angle except for a
fleeting mention.
Initial rate ¼ K3 a=ð1 þ maÞ: (6)
He also suggested that a plot of the initial rate as a
function of substrate concentration would be a
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B. Srinivasan
rectangular hyperbola [37]. Henri was supported and
advised in his effort by the work of Max Bodenstein
[41], who is considered as one of the founding fathers
of chemical kinetics [42] and the person who applied
the quasi-steady-state approximation in computing his
rate equations.
As has been pointed out earlier, Emile Fischer’s
1894 work on the lock and key model of enzyme–sub-
strate interaction was quite influential in shaping the
whole discourse in modelling the rate of enzyme kinet-
ics. Both Victor Henri and Max Bodenstein were likely
influenced by Fischer’s work. Donald Van Slyke spent
a year in the laboratory of Emil Fischer. This led him
and G.E. Cullen to propose that the binding step
between substrate and enzyme is irreversible and the
application of Bodenstein’s quasi-steady-state approxi-
mation to the derivation of their equation (irreversibil-
ity precludes rapid-equilibrium assumption) [17].
However, it was Briggs and Haldane who subsequently
showed that this approximation could be applied with
equal likelihood to reversible enzyme–substrate inter-
actions [18,43]. Having said that, the most direct influ-
ence of Fischer, who taught at the Friedrich Wilhelm
University (now called Humboldt University), would
have been on Michaelis, who studied at the university.
Three years before Michaelis and Menten published
their now famous equation, Archibald Vivian Hill,
working at the University of Cambridge, published an
equation which is now popularly known as the Hill
equation [44] (Table 1).
f ¼ Kxh =ð1 þ Kxh Þ, (7)
where, f is fractional saturation, x is oxygen tension, K
is equilibrium constant and h is the hill’s coefficient
and need not be an integer. This equation is more
complex than the MM equation.
Building upon the works of the numerous players
described above, Michaelis and Menten embarked upon
the description of the reaction catalysed by invertase.
They agreed with most of the modelling that Henri
attempted while having substantive reservations.
Mostly, Henri’s approach on the equation was criticized
by Michaelis and Menten on two fronts. Henri did not
imply pH explicitly in his treatment, likely because it
was not a prevalent idea then. It was Søren Peter Lau-
ritz Sǿrenson in 1909 and Michaelis and Davidsohn in
1911 who demonstrated the effect of pH on enzyme
activity. This work of Michaelis is what is presumed to
have drawn the interest of Menten to come over to Ber-
lin to study with him. This was because she has already
been working on the blood concentration of H+ ions in
relation to their effect on acid–base balance during
anaesthetic procedures in Cleveland with George Crile.

B. Srinivasan
This expertise of Michaelis and Menten is reflected in
their comparison of the MM equation with the dissocia-
tion equation of weak acids (Restdissoziationskurve).
Secondly, Henri did not consider the Mutarotation
of the product, which is the spontaneous (nonenzy-
matic) conversion of the ring-shaped α-D-glucose into
an equilibrium mixture with its stereoisomer β-D-
glucose [1,45]. This was highlighted by Michaelis and
Menten as another big shortcoming of the work by
Henri and was accounted for in their own work on
invertase. Further, though Henri did discuss conditions
of initial velocity, it was Michaelis and Menten who
first applied it explicitly in their treatment of the reac-
tion catalysed by invertase. Michaelis and Menten
were also the first to describe the parameter Ks as an
equilibrium constant indicative of the affinity of sub-
strate for the enzyme.
Subsequently, G.E. Briggs and J.B.S. Haldane
derived the equation with steady-state assumptions
and this is the version that we popularly know as the
MM equation (Table 1).
Steady-state treatment and its
limitation
As shown earlier, the MM equation and its derivation
are based on a plethora of assumptions that can con-
strain the interpretation of the equilibrium and rate
constants derived from them (Table 2). The principal
limitation of steady-state enzyme kinetics is the com-
plexity in interpreting the parameters Km and kcat.
Notwithstanding the strangeness of that statement, the
parameters are a composite function of several events
(and individual rate constants) happening at the
enzyme’s active site and their comprehension is condi-
tional on how the initial model is defined. For
instance, Scheme 1 would yield us the following
expressions for Km and kcat, respectively.
Km ¼ ðk2 þ k1 Þ=k1 ; kcat ¼ k2 :
A slightly more realistic yet still minimalistic model
(Scheme 2) would give us the following definition for
the kinetic parameters.
Scheme 2
Km ¼ ðk2 k3 þ k1 k2 þ k1 k3 Þ=k1 ðk2 þ k2 þ k3 Þ;
kcat ¼ k2 k3 =ðk2 þ k2 þ k3 Þ:
As can be appreciated, even with this minimalistic
model, the number of rate constants defining the
kinetic parameters is increasing dramatically and the
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Enzyme kinetics and the Michaelis–Menten equation
meaning of Km and kcat is becoming less clear. If an
additional intermediate is specified in the model
(Scheme 3), a highly likely situation for a lot of chemi-
cal transformations, the parameters become unwieldy.
Scheme 3
Km ¼ ðk1 ðk2 ðk3 þ k4 Þ þ k3 k4 Þ þ k2 k3 k4 Þ=
k1 ððk2 þ k2 Þðk3 þ k4 Þ þ k2 k3 þ k4 k3 Þ;
kcat ¼ k2 k3 k4 =ððk2 þ k2 Þðk3 þ k4 Þ þ k2 k3 þ k4 k3 Þ
The fact that the kinetic parameters can assume such
complex forms may come as surprise to many students
who have been used to the repeated use of Scheme 1 to
teach the meaning of these parameters. In spite of the
complexity described above, steady-state kinetic param-
eters help by defining the lower limit for the value of kcat
and kcat/Km. For further details on this, the reader is
referred to exceptional reviews and textbooks authored
by Prof Kenneth A. Johnson [46,47].
Kinetic isotope effects and
magnifying the information content of
the Michaelis–Menten equation
Another important advancement which, in combina-
tion with steady-state and pre–steady-state kinetic
measurements, has yielded rich insights into enzyme
kinetic mechanisms is the kinetic isotope effect (KIE)
[48]. KIEs arise when an atom in the reactant/sub-
strate or the solvent is replaced by its isotope. KIEs
can be primary (the bond connecting the isotopic atom
is formed/broken) or secondary (where there is a
change in the bonding but not directly to the bond
connecting the isotopic atom). Further, solvent isotope
effects can be seen by replacing protium oxide (water,
HOH) with deuterium oxide (heavy water, DOD)
either completely or ratiometrically (Proton inventory
studies) [49]. Both of the above-mentioned effects can
be either normal or inverse depending on whether the
rate is slower (KIE > 1) or faster (KIE < 1) in the
presence of the heavy isotope vis-à-vis the normal one.
KIE represents a powerful tool that has already had
an immense impact on our understanding of enzyme
kinetics, dynamics and mechanism. The use of KIE in
deducing enzyme kinetic mechanism, order of sub-
strate addition, identification of the rate-limiting step,
inferring intermediates, enzyme dynamics and proton
tunnelling effect has been extensively discussed [48,50–
53], and the interested reader is requested to refer to
these resources.
6095

Enzyme kinetics and the Michaelis–Menten equation
Looking ahead and moving beyond the
steady state: Pre–steady state and
single-turnover experiments
As I had alluded to in the above sections, Scheme 1
is a very inadequate representation of the events hap-
pening at the enzyme’s active site and is a gross
approximation. Further, the model proposed by
Michaelis and Menten is limiting in describing the
catalytic step, in terms of the microscopic rate con-
stants, adequately. Thus, the steady-state approach,
though ingenious in the broader perspective of under-
standing the kinetics of an enzyme observed under
specifically constrained set of experimental conditions
and providing a comparative benchmark for compar-
ing across enzymes, is characterized by the paucity of
information as far as catalysis and information con-
tent on the rate constants is concerned. This is not to
say that steady-state treatment is without any merit.
In fact, in the absence of a concrete notion about
enzymes as proteins and the exact nature of enzyme–
substrate complex, the steady-state approach and the
MM model were able to predict the existence of the
ES complex 30 years before its existence was con-
firmed by Britton Chance using stopped-flow tech-
niques [54–57]. Further, the design of pre–steady state
and single-turnover kinetic measurements are contin-
gent upon the lower limits for kcat and kcat/Km esti-
mated from steady-state measurements. Having said
that, investigators around the world are appreciating
that they ought to move beyond the steady-state
treatment to gain novel insights into the true nature
of an enzyme’s functionality in terms of understand-
ing catalysis, appreciating the presence (or lack
thereof) of intermediates and gaining traction on the
individual rate constants [58]. Thus, modern kinetic
analysis, alongside steady-state approaches, requires
the use of fast kinetic methods that are capable of
resolving events that happen on the timescale of a
single catalytic turnover at an enzyme’s active site.
This involves stopped and quench-flow measurements
to estimate transient kinetics at extremely small time-
scales and to study intermediates in single-turnover
experiments.
A brief introduction here would attempt setting the
stage for the other approaches and the unique infor-
mation that can be obtained from them. Kinetic
approaches to study the kinetics and catalysis of an
enzyme can be broadly classed into three distinct
types: (a) steady-state or quasi-steady-state conditions
(as exemplified by the Michaelis–Menten approach
and the full progress curve global analysis), (b) pre–
steady-state conditions (to capture the events
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B. Srinivasan
happening before and during the approach to steady
state) and (c) single-turnover kinetics. The fundamen-
tal difference between the various approaches enumer-
ated above is the enzyme concentration and the ratio
of [enzyme]/[substrate] in the reaction vial. As dis-
cussed earlier, steady-state (or more commonly quasi-
steady-state) measurements typically involve monitor-
ing of multiple turnovers under conditions where the
concentration of enzyme is multiple orders of magni-
tude lower than the substrate. Under such conditions,
the time courses are usually linear under conditions
of initial velocity and the initial phase approaching
the steady state (burst phase or initial transient) is
usually not observed. This is because low enzyme con-
centration leads to miniscule quantity of stoichiomet-
ric (burst amplitude is equal to enzyme concentration)
product formation that may be below the signal/noise
ratio of the assay or might have been missed because
of the extremely small timescales. This issue can be
resolved by employing higher enzyme concentration
or by having a highly sensitive assay that can measure
at extremely small timescales. If these approaches lead
to substantial signal to noise ratio to enable reliable
detection, the intercept of the progress curve on the
ordinate (y-axis) should yield us the magnitude of the
burst (and thus the concentration of the kinetically
competent fraction of the enzyme for stoichiometric
substrate to product conversion). Moreover, when
monitoring product formation, appearance of burst is
indicative of rate-limiting product release and the rate
of that can be obtained by estimating the slope of the
steady-state. However, to measure events happening
at such small timescales, manual initiation and
quenching of reactions could prove unreliable and
reliance on rapid mixing and quenching apparatus
might be required. Hence, pre–steady-state and single-
turnover kinetics rely on stopped-flow and quench-
flow instruments that have capabilities for simultane-
ous initiation and monitoring of the reaction. Pre–
steady-state kinetics aspiring to observe burst phase
would still use high substrate/enzyme ratio while
single-turnover kinetics aspiring to look into the
active site and monitor intermediates will use an
excess of enzyme over the substrate to monitor
‘single-turnover’ as a single-exponential progress
curve. This high enzyme/substrate ratio can also help
in preventing catalytic cycling which occurs when the
magnitudes of rates for the chemical step and the
product release step are similar. In this case, excess
enzyme limits the bound substrate to a single turn-
over. Thus, the chemical step of the reaction can be
isolated and determined accurately as the first-order
rate constant (kobs).

B. Srinivasan
Conclusion and perspective
There are always discoveries, monographs and
speeches that are heralded as pathbreaking and change
the direction for a particular sphere of human activity.
The equation given to us by Leonor Michaelis and
Maud Menten is one such innovation that helped
shape the field of biochemistry in general and enzy-
mology in particular. This guide was a comprehensive
attempt at introducing it from its various different
aspects without taking the ‘textbook’ approach. The
guide dwelt on the mathematical elegance of the equa-
tion, the assumptions and approximation that went
into its derivation, the historical backdrop, the limita-
tions and how the resultant evolution of pre–steady
state and single-turnover methods have ensued. This
exercise was undertaken with the aim of instilling an
appreciation of the MM equation and its overall influ-
ence in the development of modern enzymology and
biochemistry.
Acknowledgements
I am thankful to the FEBS Journal and Prof Seamus
Martin for hosting this series and to Rachel Grimley,
Maria Flocco and James Robinson for their constant
support and enthusiasm for this series. I am also
indebted to Athel Cornish-Bowden, Judith Klinman
and Kenneth Johnson for going through the manu-
script ‘Words of advice: teaching enzyme kinetics’ and
writing to me about aspects that would benefit from
further clarifications. Those clarifications have been
incorporated as part of this ‘Guide to’ series. I am also
thankful to all my enzymologist friends across the
academia-industry spectrum who felt that a series of
assays would result in popularization of enzymology
and benefit the pedagogy of the subject. Matthew
Jackson, Matthew Bilyard and Xiang Zhai are
acknowledged for their inputs on the manuscript that
enabled its improvement.
Conflict of interest
The author declares no conflict of interest.
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