Reprinted from New Beer in an Old Bottle: Eduard Buchner and the

Growth of Biochemical Knowledge, pp. 127–133, ed. A. Cornish-Bowden,

Universitat de València, Valencia, Spain, 1997

A BRIEF HISTORY OF ENZYME KINETICS

Keith J. Laidler

Unlike a well-known scientific book with a title that begins with the
same three words, the present article really is brief and really is a
history — it actually gives a few dates. In the space available I can do
no more than give a broad outline of the development of enzyme
kinetics. To give a more detailed account a good deal of mathematics
would be necessary. This article will include no mathematical equa-
tions, and only a few simple chemical ones.
Every science has two distinct but closely-related aspects: struc-
ture, and speed. Fermentation, with which Buchner’s name is firmly
associated and which has inspired the present volume, provides a
simple example. To understand fermentation properly we need to
have knowledge of the structures of the chemical substances involved,
and of the speeds with which they undergo chemical reaction. Fer-
mentation is an example of a vast group of chemical reactions that are
catalysed by enzymes, all of which are protein molecules. The word
catalyst was coined in 1836 by the great Swedish chemist Jöns Jakob
Berzelius, who collected a number of examples of catalysis, though, as
noted in the previous chapter of this book (pp. 117–120), he did not
discover the concept of catalysis. Almost all of the chemical processes
that occur in biological systems would occur extremely slowly if
suitable enzymes were not present to speed them up; life as we know it
would be impossible.
The matter of the speed with which processes occur is of para-
mount importance as far as living systems are concerned — indeed
as far as the continued existence of the universe is concerned. The
branch of chemistry which deals with the rates of chemical processes
is known as chemical kinetics, or as chemical dynamics. The
branch that deals with the question of whether processes can occur at
all is known as chemical thermodynamics. The laws of thermo-

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dynamics allow a vast number of chemical reactions to occur, but if

there were no restrictions on them the universe as we now know it
would exist for no more than a fraction of a second. The restrictions
that exist take the form of energy barriers, which chemists call activa-
tion energies. If a chemical reaction had zero activation energy, every
time two reactant molecules came together there would be instant
reaction. If this were true for every possible chemical reaction, there
would at once be chaos. Suppose that the world were as it now is, and
the barriers were all removed. Every tree would at once react with the
oxygen of the atmosphere, and the same would happen to our bodies.
In other words, the universe as we know it is as much controlled by
the laws of chemical dynamics as by the laws of chemical thermo-
dynamics.
Chemists have been making a serious study of the rates of reac-
tions, and the factors that control them, since the middle of the 19th
century. Often they found that the rates were proportional to the
concentrations of the substances that were reacting together. In 1892,
however, the British chemist Adrian John Brown, found in a study of
the rate of fermentation of sucrose in the presence of yeast that the rate
seemed to be independent of the amount of sucrose present (Brown,
1892). Later he suggested that this result could be explained if the
invertase molecules present in the yeast formed an addition complex
with the sucrose (Brown, 1902).
This was the first time that the existence of an enzyme–substrate
complex was deduced from the kinetics of an enzyme reaction. It was
not the first time the idea had been suggested. Brown himself
mentioned in his 1902 paper that the distinguished French chemist
Charles Adolph Wurtz (1880) had found that papain appears to form
an insoluble compound with fibrin prior to hydrolysis, and that Cor-
nelius O’Sullivan and Frederick William Tompson (1890) had shown
that the activity of invertase in the presence of sucrose survives a
temperature that completely destroys it if the sucrose is not present;
they regarded this as an indication of the combination of enzyme and
sucrose molecules.
Brown also made reference in his paper to the suggestion made
by the great German organic chemist Emil Fischer (Fischer, 1894)
that the specificity of enzyme action is to be explained in terms of the
precise fitting together of enzyme and substrate molecules; this is
referred to as Fischer’s lock and key hypothesis. Normally a lock can
only be operated by a given key; a slight modification to a key
usually means that the key no longer works. It is somewhat ironic
that although Fischer’s ideas became particularly fruitful when

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physical methods, such as kinetics and X-ray crystallography, were

applied to them, he himself was scornful of such methods: Wilhelm
Ostwald suggested to Fischer that physical methods would be helpful
in organic chemistry, but Fischer replied bluntly “We have no use
for your methods”.
The next important step in enzyme kinetics was made by the
German biochemist Leonor Michaelis and his Canadian assistant
Maud Leonora Menten (Michaelis and Menten, 1913). They had
observed that the effect noted by Brown — that the rate of reaction is
independent of the concentration of the substrate — is only observed at
higher concentrations of substrate. At lower concentrations the rate
becomes proportional to the concentration of substrate. To explain this
they considered the kinetic consequences of the idea that an enzyme–
substrate complex is formed. The process can be represented as

→ ES → E + products
E+S←

where E is the enzyme molecule, S the substrate molecule, and ES the

complex. The idea of Michaelis and Menten was that an equilibrium
is established between E, S and ES, and that the slow step is the break-
down of ES. Since usually there are many more molecules of
substrate present than of enzyme (on account of the high molecular
mass of the enzyme), the enzyme becomes saturated with substrate at
higher substrate concentrations. The concentration of ES will there-
fore remain the same, and the rate will remain the same, as the
concentration of substrate is varied. At low substrate concentrations, on
the other hand, the enzyme will not be saturated, and the concentra-
tion of ES, and therefore the rate of reaction, will be proportional to the
concentration of S.
This reasoning led to the well-known Michaelis–Menten equa-
tion, which contains a constant known as the Michaelis constant.
This constant is defined in such a way that a high value means that
there is only weak binding between the enzyme and the substrate; a
low value means strong binding.
Some years later a more general formulation of the Michaelis–
Menten equation was given by George Edward Briggs and the British
geneticist John Burdon Sanderson Haldane (Briggs and Haldane,
1925). They pointed out that the Michaelis assumption that an equi-
librium exists between E, S, and ES is not always justified, and should
be replaced by the assumption that ES is present not necessarily at
equilibrium but in a steady state. The resulting equation is of the same

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form, but the Michaelis constant has a different significance with

respect to the different rate constants.
Michaelis and his colleagues also made important contributions
to our understanding of the way in which the rate of an enzyme-
catalysed reaction is affected by the pH of the solution. For some time
it had been noticed in many enzyme systems that the rate is low if
the pH is high or low, and passes through a maximum at some inter-
mediate value, which is usually not far from neutrality (pH 7). In
1911, two years before the formulation of the Michaelis–Menten equa-
tion, Michaelis and Davidsohn had concluded from this pH be-
haviour that the catalytic centres of enzymes must involve two ioniz-
ing groups (Michaelis and Davidsohn, 1911). For effective catalytic
action one of these must be in the form in which it is capable of accep-
ting a proton, while the other must be in a position to donate a proton.
Over the years this basic idea was extended, particularly to take into
account the ionization of the enzyme-substrate complex as well as that
of the enzyme (Michaelis and Rothstein, 1920; von Euler, Josephson
and Myrbäck, 1924; Waley, 1953; Laidler, 1955). A strong body of
evidence shows that the powerful catalytic activity of enzymes is due
in part to the fact that they function by being simultaneously able to
donate a proton to, and accept another proton from, the substrate
molecule. This has been referred to as a push-pull mechanism.
The basic idea that an enzyme reaction involves an enzyme–
substrate complex as an intermediate is important, but requires much
extension and elaboration. There is now a considerable body of evid-
ence suggesting that in many cases there are two or more inter-
mediates. A reaction scheme involving two intermediates may be
written as

→ ES ←
E+S← → ES´ → E + products

where ES´ is the second intermediate. Good evidence for such a

mechanism has been obtained for the hydrolysis of acetylcholine by
the enzyme acetylcholinesterase (Krupka and Laidler, 1961). In this
system the rate goes through a maximum with increasing substrate
concentration, and the evidence suggests that this is due to the attach-
ment of substrate molecules to the second intermediate ES´.
With the purification and crystallization of proteins in the nine-
teen twenties, enzyme kinetics was able to enter a new phase. It be-
came possible to study in much more detail the interactions between
enzyme molecules and their substrates. This branch of enzyme

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kinetics has been referred to as molecular kinetics (Butler, 1941). The
frequency with which enzyme and substrate molecules react together
is controlled by two main factors, the energy barrier to reaction, and
the entropy of activation, which is the entropy change when the
activated complex is formed from the reactants. Both of these factors
provide valuable information about the molecular nature of the pro-
cesses occurring. For example, a large positive entropy of activation is
good evidence that charges are being neutralized when the process
occurs; the entropy increase is due to the release of water molecules
that had been bound by the ions. This is one example of a mecha-
nistic detail that cannot easily be obtained from structural studies,
since activated complexes do not exist long enough to be detected by
any but the most sophisticated high-speed techniques.
The first to carry out kinetic studies with a pure enzyme, trypsin,
was the British physical chemist John Alfred Valentine Butler
(Butler, 1941). Many studies of the same kind have been made, with a
variety of enzymes, and the results have contributed greatly to the
understanding of enzyme action (Laidler and Bunting, 1973).
When an enzyme and a substrate are brought together, the
steady state is usually established within a few milliseconds. As a
matter of experimental convenience, most investigations of enzyme
kinetics have been concerned with the steady state. To find out what
is occurring while the steady state is being established — during what
is called the transient phase of the reaction — requires special high-
speed techniques. Two problems have to be overcome. The first is to
bring the enzyme and substrate together rapidly (as otherwise the
reaction may be over before they are properly mixed). The second is
to make measurements within short periods of time.
The first problem may sometimes by overcome by the use of
flow methods, in which solutions are forced together very rapidly.
Suitable techniques were developed in particular by Roughton and his
co-workers (Hartridge and Roughton, 1923; Millikan, 1936). An
important variant of their method was the stopped-flow method, intro-
duced by Britton Chance (Chance 1940) and later developed further
(Gibson and Milnes, 1964). Sometimes the individual steps are too fast
for their rates to be measured by flow methods, and then relaxation
methods have to be used (Eigen, 1954ab). During more recent years
much further work has been done using high-speed techniques, and
many of the kinetic details of enzyme reactions have been worked
out. The results have led to the realization that few such reactions
appear to conform to the simple pattern first suggested by Michaelis
and Menten; other reaction steps are usually involved.

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