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Quantitative Enzymology

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12 Current Enzyme Inhibition, 2015, 11, 12-31

Quantitative Enzymology
David J. Timson*

School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre,

97 Lisburn Road, Belfast, BT9 7BL. UK
Abstract: Accurate measurement of the quantitative aspects of enzyme-catalysed reactions
is critical for a deeper understanding of their mechanisms, for their exploitation in biotech-
nology and for targeting enzymes by drug-like molecules. It is important to move beyond
basic enzyme kinetics as encapsulated in the Michaelis-Menten equation. The type and
magnitude of inhibition should be determined. Since the majority of enzyme-catalysed reac-
tions involve more than one substrate, it is critical to understand how to treat these reactions
quantitatively and how their kinetic behaviour depends on the type of mechanism occurring.
Some reactions do not conform to “standard” Michaelis-Menten treatment and exhibit phenomena such as co-
operativity. Again it is important to put these phenomena onto a quantitative basis. Similarly the treatment of
the effects of pH on enzymes is often vague and uninformative without a proper quantitative treatment. This
review brings together tools and approaches for dealing with enzymes quantitatively together with original
references for these approaches.
Keywords: Enzymes, cooperativity, Michaelis-Menten equation, multisubstrate, quantitative enzymology
enzyme inhibition.

INTRODUCTION and how drugs modulate activity) and biotechnol-

Without enzymes, there would be no life. The
vast majority of biologically important reactions
happen far too slowly to sustain life. It is enzymes,
with their substantial rate enhancements which
enable these reactions to occur on meaningful
timescales. The selectivity and specificity of en-
zymes also permit them to steer biological reac-
tions towards particular products thus enabling
metabolic pathways to convert molecules and ex-
tract energy from them. Therefore, understanding
how enzymes work is critical to our overall under-
standing of biology – and this understanding re-
quires a quantitative basis. Only by understanding
the relationship between the concentrations of re-
actants and rates can we begin to gain deep, mech-
anistic knowledge of a particular enzyme. When
coupled with structural and biochemical data, it is
possible to build up a picture of how these remark-
able catalysts work. This insight has implications
for basic science, medicine (both from understand-
ing how mutations affect enzyme function
*Address correspondence to this author at the School of Biological
Sciences, Queen’s University Belfast, Medical Biology Centre, 97
Lisburn Road, Belfast, BT9 7BL. UK; Tel: +44(0)2890975875;
Fax: +44(0)2890975877; E-mail: d.timson@qub.ac.uk
ogy (where exploiting and engineering enzymes
for use in industrial catalysis is of increasing inter-
est). Some important examples of enzymes of im-
portance or interest in these fields are listed in Ta-
ble 1.
RATE, ACTIVITY AND SPECIFIC ACTIVI-
TY
Rates of enzyme catalysed reactions are typical-
ly measured as rates of change of concentration –
either the appearance of product or the disappear-
ance of substrate (e.g. in units of μM min-1). The
activity of an enzyme refers to the amount of sub-
stance converted per unit time (e.g. μmol min-1) at a
given substrate and enzyme concentration. A more
useful quantitative measure of an enzyme’s cata-
lytic power is the specific activity. In general, this
is measured at a single substrate concentration and
a single enzyme concentration. It is typically ex-
pressed as amount of substrate transformed (or
product produced) per unit time per unit mass of
protein (e.g. μmol min-1 mg-1). These measures
have the advantage of being applicable to mixtures
of proteins (for example, cell extracts). Thus they
can be useful in comparing the enzyme activity in
different cells or tissues of an organism or the

1875-6662/15 $58.00+.00 © 2015 Bentham Science Publishers

Quantitative Enzymology Current Enzyme Inhibition, 2015, Vol. 11, No. 1 13

Table 1. Some examples of enzymes important in metabolism, disease, drug action and biotechnology.

Enzyme EC number Comments Ref.

Metabolism Hexokinase 2.7.1.1. Catalyses the phosphorylation of glucose, the first committed step in glycolysis. [50]
Bovine enzyme has random ternary complex mechanism.
Phosphofructoki- 2.7.1.11 Catalyses the phosphorylation of fructose 6-phosphate, a key step in glycolysis. [51]
nase Bacterial enzyme shows positive cooperativity towards fructose 6-phosphate.
Succinate dehy- 1.3.5.1 Mitochondrial membrane associated enzyme which catalyses a step in the tricar- [52]
drogenase boxylic acid cycle. Competitively inhibited by malonate.
Galactokinase 2.7.1.6 Catalyses the phosphorylation of galactose in the Leloir pathway. Has a variety of [37, 38,
ternary complex mechanisms, depending on the species. 40, 41,
53]
Galactose muta- 5.1.3.3 Catalyses the equilibrium between the - and -anomers of galactose and other [54]
rotase monosaccharides. Key catalytic residues have been identified by pH studies.
Disease Galactose 1- 2.7.7.12 Key enzyme in the Leloir pathway of galactose metabolism. Mutations in the [55-58]
phosphate uri- corresponding gene can result in type I galactosemia. Substituted enzyme mecha-
dylyltransferase nism.
NAD(P)H quinone 1.6.5.2 Enzyme involved in quinone metabolism and detoxification. A common poly- [59, 60]
oxidoreductase 1 morphism (p.P187S) is associated with an increased risk of cancer. NQO1 has a
(NQO1) substituted enzyme mechanism and is competitively inhibited by the anticoagu-
lant dicoumarol.
Coagulation factor 3.4.21.22 A glycosylated, extracellular serine protease enzyme required for blood clotting. [61, 62]
IXa Deficiency results in haemophilia type B which can be treated by injection of
recombinant factor IXa. Current research efforts are focused on increasing the
plasma half-life of this proteins.
-Secretase 1 3.4.23.46 Membrane-bound protease. Increased levels are associated with late-onset non- [63, 64]
(BACE 1) familial Alzheimer’s disease. Inhibition of this enzyme is possible, future treat-
ment.
Viral neuramini- 3.2.1.129 Catalyses the hydrolysis of sialic acid groups from complex oligosaccharides. [65-70]
dase (sialidase) Essential enzyme for release of some viruses from host cells. The enzyme is the
target of a number of antiviral drugs, including zanamivir (Relenza) and oselta-
mivir (Tamiflu).
Drug action Cyclooxygenase 1.14.99.1 Key enzyme of prostaglandin synthesis. Target of the non-steroidal anti- [19, 71]
inflammatory drugs aspirin (irreversible, covalent inhibition) and ibuprofen
(reversible, competitive inhibition)
Phosphoglycerate 2.7.2.3 Glycolytic enzyme. Likely target of the veterinary anthelminthic drug Clorsulon [72]
kinase which acts as a competitive inhibitor.
Phosphodiesterase 3.1.4.17 Catalyses the hydrolysis of cyclic AMP and cyclic GMP and thus responsible for [73, 74]
the down-regulation of a number of cellular processes. The human PDE5 isoform
is the target of sildenafil sulphate (Viagra) which acts as a competitive inhibitor.
Mitogen-activated 2.7.12.2 Cell signalling enzyme. The potential anti-cancer drug Selumetinib is an uncom- [75, 76]
protein kinase petitive inhibitor.
kinase (MAPKK)
-lactamase 3.5.2.6 Enzyme implicated in bacterial resistance to penicillin family antibiotics. Current [77-80]
interest in developing inhibitors of this enzyme to potentiate pencillins and over-
come resistance.
Biotechnology Laccase 1.10.3.2 Copper containing redox enzymes with biotechnological potential. Studies on the [81-83]
pH effects identified pKa values of functional groups involved in catalysis. Many
laccases show considerable thermal stability.
Glucose oxidase 1.1.3.4 Catalyses the oxygen dependent oxidation of glucose. Used for the detection of [84]
glucose concentrations (e.g. in blood glucose meters for diabetics).
Thermolysin 3.4.24.27 Thermostable protease. Reverse reaction exploited in the synthesis of short pep- [85-87]
tides, including the sweetener aspartame.
Urease 3.5.1.5 Catalyses the hydrolysis of urea to carbon dioxide and water. Potential for use in [88]
biosensors for urea.
Cellulase 3.2.1.4 Catalyses the hydrolysis of cellulose. Potential importance in optimising biofuel [89-91]
yields from plant based feedstocks. Vanillin (and some other phenolic com-
pounds commonly present in plant materials) is reported to be a non-competitive
inhibitor of some cellulases.
Readers are also referred to the Bremen Enzyme Database (BRENDA; http://www.brenda-enzymes.org/) for a comprehensive database of enzymes and their kinetic properties.
14 Current Enzyme Inhibition, 2015, Vol. 11, No. 1
amount of recombinant enzyme produced under
different culture conditions. However, their utility
is limited by the non-linear dependence of rate on
substrate concentrations under most experimental
conditions (see below). This means, that if mean-
ingful comparisons are to be made, then care must
be taken to duplicate exactly the concentrations of
reagents used.
THE MICHAELIS-MENTEN EQUATION
Leonor Michaelis and Maud Menten first de-
rived the equation (Equation 1) that bears their
names based on experimental work on invertase
and using the assumption that the enzyme and sub-
strate form an equilibrium complex prior to reac-
tion [1, 2]. Briggs and Haldane demonstrated that
this equilibrium assumption (which is unlikely to
apply to the majority of enzymes) was not neces-
sary and that essentially the same equation could
be derived using steady-state kinetic principles [3].
The equation was, therefore, likely to be applica-
ble to a wide range of single substrate enzymes
provided that certain assumptions are satisfied.
The substrate concentration must greatly exceed
that of the enzyme and the chemical step (i.e. the
conversion of substrate to product) must be the
slowest step of the reaction.
















Mathematically, this equation describes a rec-
tangular hyperbola. It gives rise to a curve (Fig.
1a) which initially rises steeply before plateauing.
Vmax is the maximum possible rate under the con-
ditions of the experiment. The rate (v) asymptoti-
cally approaches Vmax but never reaches it at any
physically achievable substrate concentration [S].
The Michaelis constant (Km) is equal to the sub-
strate concentration which gives a rate equal to
half Vmax. It thus has units of concentration. This is
often, erroneously, equated to the equilibrium con-
stant for the enzyme substrate complex. In fact,
under steady-state conditions, it is a composite of
three different rate constants (see below). Steady-
state conditions apply when the rate of enzyme-
substrate complex formation equals the rate of its
conversion to products; in other words, when the
concentration of enzyme-substrate complex is con-
stant. Thus, in a simplified mechanism for a sin-
gle-substrate enzyme, a series of steps can be de-
fined (Fig. 1b) beginning with the initial, reversi-
David J. Timson
ble formation of the enzyme-substrate complex,
the conversion of this complex to an enzyme
product complex and the (reversible) release of the
product, regenerating the pure enzyme [4].
(a)
(b)
k+1 k2
E+S ES EP E+P
k1
Fig. (1). The fundamentals of steady-state enzyme kinet-
ics with a single substrate enzyme (a) An ideal Michaelis-
Menten curve. In this case, Km = 30 μM and Vmax = 100 μMs-1
(b) A simplified kinetic mechanism for a one substrate en-
zyme catalysed reaction. E, enzyme; S, substrate; P, product.
From this, the steady state constants Km and
Vmax can be defined based on the rate constants of
(1) the individual steps (for definitions of the rate con-
stants, see Fig. 1b).








(2)
where [E] is the total, active enzyme concentra-
tion. The rate constant k2 is often referred to as kcat
or turnover number (or catalytic rate constant).
The turnover number is a measure of the number
of substrate molecules converted per enzyme mol-
ecule per unit time. In general, it has units of in-
verse time (e.g. min-1).










(3)




Thus, the Michaelis constant only approximates
to the dissociation constant for the enzyme-
substrate complex if k2 is small compared to k-1.
There is another useful constant which arises
from the Michaelis-Menten equation and that is
the ratio of kcat/Km. This is referred to as the speci-
ficity constant and can be used as a measure of
which substrate enzyme “prefers”: the higher the
Quantitative Enzymology Current Enzyme Inhibition, 2015, Vol. 11, No. 1 15

value of the specificity constant the greater the
preference of the enzyme for that substrate. It has
units of inverse concentration per second, i.e. the
same as for second order rate constants (e.g. μM-1
s-1 or l mol-1 s-1). The specificity constant can be
used to rank substrate preferences of enzymes. For
example, human galactose mutarotase has a speci-
ficity constant of 340,000 l mol-1 s-1 for galactose
and 90,000 l mol-1 s-1 for glucose; the enzyme
clearly “prefers” galactose as a substrate [5].
DETERMINING KINETIC PARAMETERS
FROM THE MICHAELIS-MENTEN EQUA-
TION
In practice, accurate estimation of Km and Vmax
from visual inspection of a plot of v against [S] is
almost impossible. Even when the substrate con-
centration is ten times the Michaelis constant, the
rate is only 0.91Vmax. Various practical issues
make it difficult to achieve even these levels of
substrate concentration in many cases. These in-
clude limited solubility of the substrate, limited
supply of the substrate and that some substrates
have other effects on the reaction (e.g. they affect
pH or ionic strength) which become apparent at
higher concentrations. Therefore, several methods
to linearise enzyme kinetic data have been pro-
posed. Of these, the most widely used is the so-
called Lineweaver-Burk (or double reciprocal) plot
in which the reciprocals of the rate and the sub-
strate concentration are plotted against each other
(Fig. 2a, b) [6, 7]. Despite its common use, this
plot suffers from some well-documented short-
comings. The most significant of these is the ten-
dency to magnify errors in small quantities. Often
the smallest rates are the hardest measure and thus
subject to the greatest errors. However, in a double
reciprocal plot, these small values become large
and often have a disproportionate effect on the fit-
ting of the straight line [7-11]. This can be partial-
ly mitigated by planning the experiment in “recip-
rocal space”, i.e. such that the points are evenly
distributed in the Lineweaver-Burk plot.
A number of alternatives to the Lineweaver-
Burke plot exist. These include the Eadie-Hofstee
plot (v against v/[S]) and the Hanes-Woolf plot
([S]/v against [S]) (Fig. 2a, c, d) [12-15]. While
these eliminate some of the issues associated with
the Lineweaver-Burk plot, they bring the new is-
sue that one variable is present on both axes. Thus,
any errors associated with the measurement of that
variable affect both the x- and y-coordinates and

(a) (b) (c)

Fig. (2). Linearisation of steady-state kinetic data (a) A plot of v against [S] showing simulated data points for which Km =
30 μM and Vmax = 100 μMs-1. (b) The same data transformed to make a Lineweaver-Burk plot. Note how the data points bunch
towards the lower left of the graph, despite being evenly spaced in (a). (c) The same data transformed to make an Eadie-
Hofstee plot. (d) The same data transformed to make a Hanes-Woolf plot. [A note on the representation of units in this and
other figures: here the convention is used to show the variable separated from the unit by a forward slash (“/”). This indicates
that the quantities shown on the axes are the result of dividing the value by the unit, resulting in a pure number. For example, if
the number 10 is shown on the axes and the axis is labelled “[S]/μM”, then this tells us that the actual value (10 μM) has been
divided by the unit (μM) leaving the pure number. This convention is used in many journals and is preferred by nomenclature
and standard setting bodies, for example the National Institute of Science and Technology (NIST, USA) [49].
16 Current Enzyme Inhibition, 2015, Vol. 11, No. 1 David J. Timson

disproportionately influence the estimate.
Increasingly, direct non-linear fitting of the ki-
netic data is used to estimate kinetic constants
from experimental data. Linear fitting (regression)
is widely used in science and many commonly
used software packages include linear regression
functions. Normally, linear regression is achieved
by the least squares method. In this method, the
“best fit” line is the one with the lowest sum of the
squares of the differences between the line and the
data points. In general, there is one unique, analyt-
ical solution to a least squares fit of any data.
(However, it should be noted that the fit will be
meaningless if the data do not conform to a linear
relationship.) Linear regression can be used in
combination with linearized kinetic data to esti-
mate kinetic constants. Non-linear fitting works by
similar principles, i.e. the minimisation of the sum
of the squares of the differences between the line
(model) and the experimentally determined data
points [16]. However, there is rarely a single, ana-
lytical solution and the “best fit” must be found by
incrementally changing the values and testing
whether they make the fit better or worse. Inevita-
bly this is more computationally demanding, espe-
cially for complex equations. Nowadays a number
of software packages are available which carry out
robust non-linear curve fitting and these can be
readily used with enzyme kinetic data. Since no
linearization is involved, the estimates obtained
are generally considered more accurate than those
determined by the Lineweaver-Burk plot and other
similar methods. However, it is necessary to avoid
the trap of assuming that the computer program
“knows best”. Any fit should be critically as-
sessed. If it clearly deviates from the data points,
either the fit is a poor estimate or the equation to
which the data are being fitted is inappropriate
(Fig. 3). Better estimates can sometimes be ob-
tained by altering the initial estimates of the pa-
rameters (since all non-linear fitting programmes
can fail to find the best fit). The appropriateness of
the equation can be tested statistically by compar-
ing it with fits to other possible equations. Another
measure of the quality of the fit comes from calcu-
lation of the residuals, that is the difference be-
tween the fit and the points. The residuals should
be small in comparison to the measured values and
randomly distributed about zero (Fig. 3). It is pos-
sible to test both of these statistically; however,
visual inspection of the residuals is often enough
to detect a poor quality fit. Ultimately, the judg-
ment lies with the experimenter and fits which are
clearly poor on inspection should be rejected re-
gardless of what the computer program says.
THE NATURE AND TYPES OF ENZYME
INHIBITION
Enzyme inhibitors are compounds which re-
duce the rate of an enzyme catalysed reaction.

(a) (b) (c)

Fig. (3). Non-linear curve fitting. (a) Simulated data fitted to the Michaelis-Menten equation (top) with small, randomly dis-
tributed residuals (bottom). (b) Simulated data fitted to the Michaelis-Menten equation (top) with large but randomly distribut-
ed residuals (bottom). (c) Simulated data fitted to the Michaelis-Menten equation (top) with small, but non-randomly distribut-
ed residuals (bottom). Note how in this case, the actual fit looks reasonable but the residuals reveal that there is a problem with
either the data or the choice of equation used for non-linear fitting.
Quantitative Enzymology
Normally, we are most interested in those com-
pounds which inhibit specific enzymes or reac-
tions. Although there are a number of compounds
which inhibit a wide range of enzymes, these nor-
mally do so through denaturation of the protein
(e.g chaotropic agents such as urea [17]). Enzymes
which target specific enzymes or groups or related
enzymes are more likely to be useful as leads in
drug discovery or reagents for use in laboratory
investigations.
Careful, quantitative measurement of the effects
of specific inhibitors on the rates of enzyme cata-
lysed reactions can reveal much about the molecu-
lar basis of their action. Enzyme inhibitors can be
divided into two main classes – reversible and ir-
reversible. Irreversible inhibitors are those which
bind so tightly to the enzyme that they cannot be
removed by common laboratory separation tech-
niques (e.g. dialysis, gel filtration chromatog-
raphy). This means that the interaction between
the enzyme and inhibitor can be detected by meth-
ods which measure changes in molecular mass, for
example, MALDI-TOF mass spectrometry. In
general, irreversible inhibitors form covalent
bonds with the enzyme molecule. Thus, stoichio-
metric amounts of inhibitor can result in total inhi-
bition of the enzyme. The effects of irreversible
inhibitors on steady state enzyme kinetics can be
hard to quantify. In addition to the enzyme-
catalysed reaction a second chemical process is
occurring, i.e. the chemical reaction between the
enzyme and the inhibitor. It is, of course, possible
to treat this enzyme-inhibitor reaction as a classi-
cal second order process and derive rate constants
for this. The widely prescribed analgesic and anti-
inflammatory drug aspirin (acetylsalicylic acid) is
an irreversible inhibitor of cyclo-oxygenase 2
(COX-2) [18-21]. Irreversible inhibitors have
some advantages as drugs. Since stoichiometric
amounts are required for inhibition, doses can be
low. Their irreversible nature means that once in-
hibited, an enzyme molecule is unlikely to regain
activity. Therefore, the only way the cell can re-
gain that activity is to synthesise more enzyme.
However, the reactive nature of some irreversible
inhibitors means that there is a potential risk of
covalent modification of non-target proteins which
could lead to significant side effects.
Reversible inhibitors normally interact with en-
zymes through weaker, non-covalent bonding.
There are various subgroups of reversible inhibi-
Current Enzyme Inhibition, 2015, Vol. 11, No. 1 17
tors and the nomenclature for these has become
confused over the years. The most commonly de-
scribed form of reversible inhibition is competitive
inhibition. Competitive inhibitors bind to enzymes
at their active sites and sterically hinder access by
the substrate. One consequence of this molecular
mechanism is that the degree is inhibition is con-
trolled by the relative concentrations of the sub-
strate and the inhibitor. Thus, the degree of inhibi-
tion can be reduced by increasing the concentra-
tion of substrate. The binding of a competitive in-
hibitor to an enzyme can be considered as an equi-
librium process with the equilibrium constant, Kic.
Since a purely competitive inhibitor cannot bind to
the enzyme-substrate complex (because its binding
site is occupied) then it must only bind to the free
enzyme. The equilibrium constant, Kic is also
known as the competitive inhibition constant and it
is defined in Equation 4.






(4)


([I] is the concentration of inhibitor and [EI] the
concentration of the non-covalent enzyme-
inhibitor complex).
Competitive inhibition is not the only type of
inhibition encountered. Uncompetitive inhibition
occurs when the inhibitor interacts with the en-
zyme-inhibitor complex at a location other than
the active site. Therefore, uncompetitive inhibi-
tors do not, directly interfere with the binding of
substrate and their effects cannot be reversed by
the addition of higher concentrations of substrate.
At a molecular level, uncompetitive inhibitors
reduce the rate of reaction due to allosteric ef-
fects: binding at one site causes changes in the
protein’s overall conformation which affects the
structure, and therefore reactivity, of the active
site. Uncompetitive inhibition is characterised by
the uncompetitive inhibition constant, Kiu; like its
counterpart in competitive inhibition this is also a
real equilibrium constant, defined according to
Equation 5:






(5)


([ES] is the concentration of the enzyme-substrate
complex; [ESI] is the concentration of the enzyme-
substrate-inhibitor complex).
It is also possible to conceive of a third type of
inhibition in which the inhibitor binds both to the
18 Current Enzyme Inhibition, 2015, Vol. 11, No. 1
free enzyme and to the enzyme-substrate complex.
Such inhibition will be characterised by both com-
petitive and uncompetitive inhibition constants and
is known as mixed inhibition. Non-competitive
inhibition is a special case of mixed inhibition in
which the inhibitor binds equally strongly to the
free enzyme and the enzyme-substrate complex
and, thus, the competitive and uncompetitive inhi-
bition constants are equal. Some authors doubt that
non-competitive inhibition occurs commonly in
real enzyme systems [22]. In reality, mixed inhibi-
tion is likely to be by far the most common type of
reversible inhibition. However, either the competi-
tive or uncompetitive element may be far greater
than the other and may thus dominate any analysis.
QUANTIFICATION OF REVERSIBLE EN-
ZYME INHIBITION
The Michaelis-Menten equation (Equation 1)
can be modified to allow for the effects of reversi-
ble inhibition [22]. Using these equations and
carefully designed experiments, the inhibition con-
stants can be determined. The steady state equa-
tion for competitive inhibition (Equation 6) modi-
fies the Km term, reflecting the fact that competi-
tive inhibition affects only Km and not Vmax.
Vmax [S]
v=
 [I] 
K m  1+ + [S]
 K ic 
This equation describes a curve of similar
shape to the Michaelis-Menten equation (i.e. a
rectangular hyperbola) in which increasing the
value of [I] while keeping all other values con-
stant shifts the curve to the right without affecting
the limiting value of v (Fig. 4a). In theory, data
can be fitted to Equation 6 by non-linear curve
fitting to yield the value of Kic (and Km and Vmax).
In practice, as equations become more complex
and the number of constants increases, non-linear
curve fitting is more likely to fail. It may also be
necessary to evaluate first which form of inhibi-
tion is occurring. (Some studies mistakenly as-
sume that inhibition is competitive since this is
the best known and most straightforward to ana-
lyse.) They key feature of competitive inhibition
is that Vmax is invariant. Therefore, in a Lin-
eweaver-Burk plot, all lines should pass through
the y-axis at the same point regardless of the con-
centration of inhibitor (Fig. 4b).
David J. Timson
If the reciprocal rate of a competitively inhibit-
ed reaction is plotted against inhibitor concentra-
tion, the result is a straight line. Repeating the pro-
cess at a second substrate concentration results in
another straight line which intersects with the first
in the (-x,y) quadrant (Fig. 4c). Lines resulting
from further substrate concentrations will also
cross at the same point and the x-value of this
point is equal to –Kic (and the y-value to 1/Vmax).
This plot (known as the Dixon plot [23]) can be
used to estimate the value of the competitive inhi-
bition constant. However, it should be noted that
intersection of these lines in the (-x,y) quadrant
does not prove competitive inhibition since the
same result will be seen with mixed inhibition [22,
24]. Of course, with real experimental data the in-
tersection of three or more lines will not be exactly
on the same point and the centroid of the intersec-
tions should be calculated in order to estimate Kic.
The Dixon plot suffers from a number of prob-
lems, not least the need to use reciprocals of the
rate leading to similar issues to the Lineweaver-
Burk plot (see above) [25].
Close examination of Equation 6 shows that it
is in the same form as the Michaelis-Menten equa-
tion. Indeed, it can be rewritten as:
Vmax [S]
(6) v= (7)
K m,app + [S]
Here, Km,app (the apparent Km) replaces one of the
terms in the denominator of the fraction such that:













(8)



Therefore, if the apparent Km is determined for
a range of different values of inhibitor concentra-
tion then a plot of Km,app against [I] can be used to
determine Kic (Fig. 4d). The gradient of the line
will be 1/Kic (and the y-intercept will be Km). This
relatively straightforward example illustrates a
general case that, where more complex equations
can be reduced to a form similar to the Michaelis-
Menten equation, it is then possible to construct
so-called secondary plots (or replots) to determine
the inhibition constants.
In uncompetitive inhibition both Km and Vmax
are both reduced by the same fraction; therefore
the ratio of Vmax to Km is unaffected by the pres-
ence of an uncompetitive inhibitor (Fig. 5a). In a
Lineweaver-Burk plot, increasing concentrations
of an uncompetitive inhibitor give rise to parallel
Quantitative Enzymology Current Enzyme Inhibition, 2015, Vol. 11, No. 1 19

(a)

(b)(i) (ii)

(c) (d)

Fig. (4). Competitive inhibition (a) Michaelis-Menten plots of simulated kinetic data with , Km=30 μM, Vmax=100 μMs-1,
Kic=10 μM and (from top curve to bottom) 0, 10, 25 and 50 μM inhibitor. (b) (i) A Lineweaver-Burk plot using the same simu-
lated data as (a). (ii) a close-up of the graph shown in (b)(i) showing the y-intercept; note how all lines pass through the same
intercept because Vmax is unaffected. (c) A Dixon plot using data for three selected substrate concentrations from (a) – one be-
low Km (16.7 μM), one at Km (30 μM) and one higher than Km (70 μM). Note how the lines intercept in the (-x,y) quadrant as
indicated by the arrow. (d) A secondary plot constructed according to Equations 7 and 8 (see text for detail) and using the same
data as in (a).

lines (since the gradient in this plot is Km/Vmax) In order to produce a form which resembles the
(Fig. 5b). Parallel lines are also seen in the Dixon Michaelis-Menten equation, it is necessary to have
plot with uncompetitive inhibitors (Fig. 5c). An [S] in the denominator not subject to multiplica-
alternative form of this plot has been proposed for tion by any term. Dividing [S] (and all other
the determination of the uncompetitive inhibition terms) by (1+[I]/Kiu) results in an alternative form
constant Kiu. In this plot [S]/v is plotted against of Equations 9:
[I] for a range of different substrate concentra-









tions. The result is very similar to the Dixon plot:




the lines intersect in the (-x,y) quadrant such that



(9a)








the x-value at the interception point is - Kiu (and





the y-value is equal to Km/Vmax) (Fig. 5d) [26].
The steady state rate equation for uncompetitive This is in the same form as the Michaelis-
inhibition is: Menten equation and can be rewritten as:
Vmax [S] Vmax, app [S]
v= (9) v= (9b)
 [I]  K m , app + [ S]
K m + [S]  1+
 K iu 
20 Current Enzyme Inhibition, 2015, Vol. 11, No. 1 David J. Timson

(a) (b)

(c) (d)

(e) (f)

Fig. (5). Uncompetitive inhibition (a) Michaelis-Menten plots of simulated kinetic data with , Km=30 μM, Vmax=100 μMs-1,
Kiu=10 μM and (from top curve to bottom) 0, 10, 25 and 50 μM inhibitor. (b) A Lineweaver-Burk plot using the same simulat-
ed data as (a). (c) A Dixon plot using data for three selected substrate concentrations from (a), –16, 32 and 64 μM. Note how
the lines are parallel in this type of inhibition. (d) A plot of [S]/v against [I] for the same three substrate concentrations shown
in (c). Note how the lines intercept in the (-x,y) quadrant as indicated by the arrow. (e) A secondary plot showing the depend-
ence of Km,app on inhibitor concentration, constructed according to Equation 10 (see text for detail) and using the same data as
in (a). (f) A secondary plot showing the dependence of Vmax,app on inhibitor concentration, constructed according to Equation
11 (see text for detail) and using the same data as in (a).

Therefore, in this case, there are two apparent f). There are a variety of approaches to lineariza-
kinetic constants, both of which are dependent on tion, but the most straightforward is to take recip-
the inhibitor concentration. These are defined as: rocals of the apparent kinetic constant and plot
these against the inhibitor concentration. This











(10) yields a plot in which the gradient is 1/KiuVmax and



the y-intercept is 1/Vmax. This is clearly somewhat


cumbersome and nonlinear curve fitting is pre-


 ferred. It is also clear from Equations 10 and 11








(11)



that the ratio of Vmax,app/Km,app will be equal to


Vmax/Km for all values of inhibitor concentration.
Both equations 10 and 11 can be used to deter- Since kcat is the maximum rate divided by the ac-
mine the uncompetitive inhibition constant. This tive enzyme concentration (Equation 2) it also fol-
can either be done by direct, nonlinear curve fit- lows from this that the specificity constant is unaf-
ting or by linearization of these equations (Fig. 5e, fected by an uncompetitive inhibitor.
Quantitative Enzymology
Mixed inhibition affects both Km and Vmax; both
constants are altered by different fractions. Thus, it
requires both a competitive and uncompetitive in-
hibition constant to describe it quantitatively. Non-
competitive inhibition can be considered as a spe-
cial case of mixed inhibition, in which Kic and Kiu
are equal. This has the effect of keeping Km con-
stant and reducing Vmax (Fig. 6). The general
steady state rate equation for non-competitive and
mixed inhibition is:
Fig. (6). Noncompetitive inhibition is a special case of
mixed inhibition. Michaelis-Menten plots of simulated ki-
netic data with Km=30 μM, Vmax=100 μMs-1, Kic=Kiu=10 μM
and (from top curve to bottom) 0, 10, 25 and 50 μM inhibi-
tor. In noncompetitive inhibition, Km remains constant and
Vmax is reduced.
Vmax [S] (12)
v=
 [I]   [I] 
K m  1+  + [S]  1+
 K ic   K iu 
Equation 12 can be reduced to a form similar to
the Michaelis-Menten equation by a similar ap-
proach to that adopted for Equation 9, i.e. dividing
through by (1+[I]/Kiu). The definition of the appar-
ent maximum rate, Vmax,app is the same as for un-
competitive inhibition (Equation 11) and the ap-
parent Michaelis constant, Km,app is:



















(13)





There is no straightforward linearization of
Equation 13. However, if Kiu has already been de-
termined it is possible, although cumbersome, to
determine Kic from a plot of Km,app(1+[I]/Kiu)
against [I]. Once again, nonlinear curve fitting is
preferable; in the case of Equation 13 it can be
Current Enzyme Inhibition, 2015, Vol. 11, No. 1 21
simplified by fixing the values of Km and/or Kiu if
these have already been determined.
The approach of simplifying rate equations so
that they are in the form of the Michaelis-Menten
equation and then deriving equations for the ap-
parent kinetic constants is applicable to competi-
tive, uncompetitive and mixed inhibition. In each
case, one or more secondary plots are required in
which apparent kinetic constants are plotted
against the inhibitor concentration. This requires
the apparent constants to be known for a range of
inhibitor concentrations. Although the secondary
plot method is generally considered to be the “gold
standard” for determining inhibition constants,
there are sometimes practical issues. Probably the
most critical is a lack of time or reagents to per-
form the many individual assays required. In these
cases, it may be necessary to resort either to non-
linear fitting of the appropriate rate equation at one
inhibitor concentration or the use of Dixon plots
and their variants.
An alternative quantitative measure of inhibi-
tion is the concentration of inhibitor required to
result in a 50% reduction in the rate (IC50). This
parameter is widely used for the screening of in-
hibitors in the pharmaceutical and other industries.
Often it is determined by using a wide range (sev-
eral orders of magnitude) of inhibitor concentra-
tion and so this is often plotted on a logarithmic
scale (Fig. 7a). It has the advantage of being rela-
tively straightforward to determine and interpret;
IC50 is particularly useful for compiling rankings
of the inhibitory potential of large numbers of
compounds. However, it provides no information
on the underlying type of inhibition. Such infor-
mation may be useful if further development of an
inhibitor is required since the type of inhibition
provides some information on how the compound
interacts with the enzyme. Another issue is that
IC50 values are highly dependent on the substrate
concentration used in the assay – and how these
two parameters are related depends on the type of
inhibition. For example, in competitive inhibition
a much greater degree of rate reduction is ob-
served when the substrate concentration is close to
Km than when it is many times Km and the rate is
approaching Vmax; therefore a much higher inhibi-
tor concentration is required to give a 50% reduc-
tion in rate (Fig. 7b). Considerable care should be
taken when comparing IC50 values from different
laboratories since the conditions of the assay (most
22 Current Enzyme Inhibition, 2015, Vol. 11, No. 1
(a)
(b)
Fig. (7). IC50 is an alternative parameter to quantify in-
hibition. (a) A typical inhibition curve used to estimate IC50.
In this case, the enzyme is undergoing competitive inhibition
with the simulated parameters Km=30 μM, Vmax=100 μMs-1
and Kic=10 μM; the substrate concentration is equal to the
Km. (b) The effect of increasing [S] on IC50. Parameters are
the same as in (a), expect for [S] which was varied as shown.
Fitted IC50 values were 12, 20, 60, 100 μM for [S]=5, 30,
150, 300 μM respectively. In both (a) and (b), v0 represents
the initial rate in the absence of inhibitor.
critically, the value of [S]) are unlikely to be simi-
lar. If the type of inhibition, the Michaelis constant
and substrate concentration is known, IC50 values
can be converted to inhibition constants using the
Cheng-Prusoff relationships [27]. For example,
with competitive inhibitors, the relationship is:









(14)






Note that, in the case of competitive inhibition,
when the substrate concentration is equal to Km,
this simplifies to Kic=IC50/2.
ENZYME-CATALYSED REACTIONS WITH
MORE THAN ONE SUBSTRATE
The majority of enzymes catalyse reactions in-
volving two or more substrates. However, much of
David J. Timson
the literature on the theoretical basis of quantita-
tive enzyme kinetics concentrates on reactions
which involve the conversion of one substrate to
one product. (The most common examples of the-
se are isomerisations; some reactions in which one
reactant is in vast excess, e.g. hydrolytic reactions,
can also be treated as being single substrate reac-
tions). Once we consider the general reaction in
which two substrates are converted to two prod-
ucts, the steady state rate equations need to be
modified to account for this. A general form of a
two substrate enzyme-catalysed reaction can be
written as:
A + B C + D
in which A and B are substrates and C and D are
products. There are several different ways in
which A and B can interact with the enzyme – and
there are different steady-state rate equations de-
pending on how this occurs [28-31].
In the substituted enzyme mechanism, A first
interacts with the enzyme and reacts with it. This
reaction normally involves the transfer of a chemi-
cal group or electrons to the enzyme (or to a coen-
zyme strongly associated with the enzyme). Ex-
amples of commonly transferred groups include
hydrides and phosphates. Since the enzyme has
been covalently modified by reaction with A, it
should be possible to detect this modification by
analytical techniques. For example, if A transfers a
phosphate group to the enzyme then there would
be an increase in mass of the protein, detectable by
mass spectrometry (and other methods which
measure molecular mass). Alternatively if A trans-
fers electrons to a coenzyme, this reduction is like-
ly to be detectable spectrophotometrically. Once A
has reacted with the enzyme, the remainder of the
molecule it will diffuse away from the active site
as product C. Therefore, reaction of enzyme with
A (in the absence of B) can be treated as a second
order chemical process and, often, exposing the
enzyme to an excess of A will convert the vast ma-
jority of enzyme molecules. The enzyme then re-
acts with B, transferring the group which came
from A to B. In this second step of the reaction,
the enzyme is regenerated and product D is
formed. One feature of this mechanism is that A
and B do not, directly, react with each other. In-
deed, they do not occupy the active site at the
same time (Fig. 8a). This kinetic mechanism is
sometimes known as the “ping-pong” reaction.
Quantitative Enzymology Current Enzyme Inhibition, 2015, Vol. 11, No. 1 23

Fig. (8). Kinetic mechanisms for enzymes with more than one substrate. (a) A schematic representation of a substituted
enzyme (or “ping-pong”) mechanism. The enzyme (E) first reacts with substrate A to form an adduct with part of A (A’), re-
leasing the first product, C. The enzyme adduct (EA’) then reacts with the second substrate, B generating the second product D
which is then released. (b) A hierarchical classification of the main mechanisms of bisubstrate enzyme catalysed reactions.

The steady state rate equation for the substitut- This yields equations for the apparent kinetic
ed enzyme mechanism is: parameters:





v=
Vmax [ A][B]
(15)










(16)




K m , B [ A] + K m, A [ B] + [A][B]





Although there is only one Vmax value for this










(17)









mechanism, each substrate has its own Michaelis
constant, Km,A and Km,B. If the concentration of A Both Equation 16 and Equation 17 are in the
is varied and the concentration of B is held con- same form as the Michaelis-Menten equation and
stant the dependence of v on [A] will be a rectan- so values for the kinetic constants can be extracted
gular hyperbola, just like the Michaelis-Menten by similar methods to those described above.
equation. This suggests a way to obtain the three Since the denominators of these two equations are
steady state kinetic parameters associated with this identical it follows that, as for uncompetitive in-
reaction. If the constant terms (i.e. Vmax, Km,A, Km,B hibitors, in the substituted enzyme mechanism
and, in this case, [B]) are collected together, Equa- Vmax,app/Km,app will be equal to Vmax/Km, i.e. the
tion 15 can be rewritten as: specificity constant is invariable.
(Vmax [B])[A] The other common kinetic mechanism for two
v= (15a)
K m,A [B] + [ A](K m,B + [B]) substrate reactions is the ternary complex mecha-
nism. In this mechanism both substrates bind to
Isolation of [A] in the denominator requires di- the active site at the same time to form a ternary
vision through by the constant term (Km,B+[B]) substrate complex (EAB). Once this has formed,
and gives a version of Equation 15 in the form of catalysis occurs and a ternary product complex
the Michaelis-Menten equation: (ECD) if formed. The products then dissociate
from the active site and diffuse away. Two types
 (Vmax [B]) 
  [A] of ternary complex mechanism can be distin-
(K + [B])
v=
m,B (15b) guished – random and ordered. In a random ter-
 K m,A [B] 
 + [ A] nary complex mechanism, the substrates can bind
(K + [B])
m,B
in any order. In contrast, in ordered mechanisms,
24 Current Enzyme Inhibition, 2015, Vol. 11, No. 1
one substrate is required to bind before the second
one. The most common, molecular explanations
for ordered mechanisms are that the first substrate
induces a conformational change in the enzyme
enabling binding to the second substrate or that the
first substrate forms part of the binding site for the
second substrate.
The steady state rate equation for both ternary
complex mechanisms is:



































The new term in this equation, KIA is hard to in-
terpret and its precise meaning varies depending
on the type of ternary complex mechanism. Equa-
tion 18 can be reduced to a form similar to the
Michaelis-Menten equation by a similar process to
that used for the substituted enzyme mechanism
(Equation 15). Once again, if one substrate con-
centration is held constant and the other varied,
constant terms can be collected together. This
yields the same equation for Vmax,app as for the sub-
stituted enzyme mechanism (Equation 16) and the
following equation for Km,app:































Equation 19 describes a rectangular hyperbola
which intersects the y-axis at KIA.
Since the two types of ternary complex mecha-
nism share a common rate equation, they cannot
be distinguished by the dependence of the initial
rate on substrate concentration. One way to diag-
nose the type of mechanism occurring is through
studying the effects of product inhibition [32-35].
Specifically, the type of inhibition (i.e. competi-
tive, uncompetitive or mixed) of both C and D
must be determined. In the random mechanism,
both C and D will be competitive inhibitors with
respect to both substrates. However, in the ordered
mechanism, where A is the first substrate to bind
and C is the first product to be released, C will be
a mixed inhibitor with respect to A and B. In con-
trast D will be a competitive inhibitor with respect
to A, but show mixed inhibition with respect to B
[33, 35]. Enzymes which have substituted enzyme
or ordered ternary complex mechanisms may also
exhibit the phenomenon of substrate inhibition.
High concentrations of the second substrate to
bind may act as inhibitors with respect to the first
substrate [32, 36].
David J. Timson
In practice examples of purely random or pure-
ly ordered mechanisms may be rare. A truly ran-
dom mechanism requires that the enzyme’s inter-
action with each substrate has no effect on the af-
finity for the other substrate. Given the mutual
structural changes which generally occur on en-
zyme-substrate binding, this is unlikely. A truly
ordered mechanism requires that the enzyme has
no affinity for the second substrate to bind (or that
it is only able to bind it in a non-productive way).
Again this is unlikely when dealing with real en-
(18) zymes. Nevertheless, well documented examples
exist of enzymes which conform (largely) to one
mechanism or the other (for example, galactoki-
nase exhibits different mechanisms, depending on
the species [37-41]).
EFFECTS OF pH ON ENZYME ACTIVITY
Hydrogen ion concentration has a variety of in-
terrelated effects on enzymes. At extremes of pH,
the majority of enzymes will be denatured and,
therefore, lose activity. However, the protonation
and deprotonation of amino acid residues can also
have more subtle effects on protein structure and
on the reactivity of active site residues. Measuring
(19) the effects of pH on enzyme activity is commonly
reported in the literature. Unfortunately, much of
what is reported is of limited value. Measurement
of the rate (or activity) at one substrate concentra-
tion over a range of pH values is not very informa-
tive. Although this experiment commonly gives
rise to a bell-shaped curve and permits the identi-
fication of an “optimum pH”, the effects observed
often combine denaturation, structural changes and
alterations in the reactivity of key active site resi-
dues. Furthermore, alteration of the substrate con-
centration may well alter the precise shape of the
curve or change the “optimum pH”.
More information rich experiments are those in
which the effect of pH on individual kinetic pa-
rameters is measured; the most useful parameters
to study are the turnover number, kcat and the spec-
ificity constant, kcat/Km. Careful measurements of
these effects can reveal some information about
the residues involved in binding and catalysis. As-
suming that there is no denaturation or other gross
structural changes, these experiments can be used
to estimate the pKa values of key groups in the ac-
tive site. In effect a titration experiment is being
carried out and the ideal result is a sigmoidal curve
in which the x-value at the inflexion point(s) cor-
Quantitative Enzymology
responds to the pKa of the functional group being
titrated; if there are two of these and they are well-
separated then the result will be a bell-shaped
curve (Fig. 9). Since denaturation gives rise to a
similar shaped curve it is important to provide ex-
perimental evidence that this is not occurring. One
simple test is to observe the solution and note the
presence or absence of any precipitates resulting
from pH-dependent denaturation. Fluorescence or
circular dichroism spectra taken at pH values
across the range tested should be similar indicating
no major structural changes. For pH values which
result in very low rates, adjustment of the pH to
more optimal values should rapidly restore the
rate.
In the ideal case, the effect of hydrogen ion
concentration on enzyme activity can be described
as:

















(20)












(Ka1 and Ka2 are the two acid dissociation con-
stants; kcat’’ is the value of kcat determined at a giv-
en pH value and kcat,lim is the limiting value of kcat)
Equation 19 assumes that there are two independ-
ent ionisable groups and that no denaturation oc-
curs in the pH range studied [42, 43].
Even if the experiment is done carefully and
there is no evidence of denaturation, care is still
required in the interpretation of the results. Under
these circumstances, the pKa values obtained will
correspond to important groups in the catalytic
process. However, assigning these to specific resi-
Fig. (9). The effects of pH on enzyme activity. A typical, ideal pKa curve in which there are two ionisable groups (pKa1=4.5
and pKa1=8.5) and no denaturation or substrate ionisation effects. The line was fitted to equation 20.
Current Enzyme Inhibition, 2015, Vol. 11, No. 1 25
dues can be difficult or even impossible. Most pro-
teins contain many groups with similar pKa values
(e.g. asparate, glutamate and the C-terminus all
have similar values) and, anyway, the pKa of a res-
idue is highly sensitive to its local environment,
including other nearby amino acid residues. In-
deed, the active sites of some enzymes are struc-
tured so that the pKa of catalytically important res-
idues are displaced substantially for the value
measured for the same amino acid in solution. Fur-
thermore, in some cases the substrate can also be
protonated and deprotonated over the pH range
being tested and, therefore, the observed effects
may reflect changes to the substrate rather than the
enzyme. ATP (and other nucleotide phosphates) is
one important group of molecules which alter their
ionisation state in the pH range 1-14 [43].
EFFECTS OF TEMPERATURE ON EN-
ZYME ACTIVITY
The effects of temperature on enzyme activity
are another area where many published experi-
ments are not very information rich. Again the is-
sue is confused by the various effects that tem-
perature can have on enzyme structure and func-
tion. In general terms, increasing the temperature
increases the amount of energy being supplied to a
reaction which tends to increase the rate. However,
protein molecules are often only marginally stable
at physiological temperatures and so, when raised
significantly above these, tend to denature. How-
ever, careful measurement of temperature-
dependent effects on activity within the range
where only catalysis (and not overall structure) is
26 Current Enzyme Inhibition, 2015, Vol. 11, No. 1 David J. Timson

affected can reveal information about the activa- (a)

tion energy of the enzyme-catalysed reaction.
Since enzymes function by reducing the activation
energy of the reaction, this is a valuable parameter
to know. It can particularly useful in helping to
rationalise why an enzyme is more efficient at cat-
alysing reactions with one substrate compared to
another.
In principle, enzyme-catalysed reactions can be
considered using the Arrhenius equation:











(21) (b)
(A is the Arrhenius constant; Ea is the activation
energy; R is the molar gas constant; T the absolute
temperature). Here, kcat’ is the kcat measured at any
given temperature and is often the most useful
constant to subject to this treatment since it reports
on the rate-determining step which is often the
chemistry of the enzyme catalysed reaction. Val-
ues for the constants can be extracted using a line-
arised form of the equation:














(21a)
Fig. (10). The effects of temperature on enzyme activity.
Therefore, a plot of the logarithm of kcat’ (a) A typical, idealised Arrhenius plot showing the depend-
against 1/T will yield a straight line with negative ence of kcat on temperature assuming no denaturation or oth-
slope (Fig. 10). The gradient will be –Ea/R (and er gross structural changes occur in the temperature range
the y-intercept will be the logarithm of A). studied. Data were simulated using Equation 20 with the
parameters A=10,000 s-1 and Ea=20 kJmol-1. (b) Linearisa-
COOPERATIVITY tion of the data shown in (a) according to Equation 21a.

Not all enzymes conform to the Michaelis-
Menten equation. One common reason for this is
the phenomenon of cooperativity. This was first
well-characterised in the non-enzymatic protein
haemoglobin where it was observed that the bind-
ing of oxygen molecules to the four binding sites
became progressively easier as the sites were filled
[44]. In other words, binding of oxygen at one site
in some way affects the other sites to increase their
affinity for the molecule. The same process can be
observed in many (but not all) enzymes which
have more than one functional active site in the
same protein. Cooperativity requires that there is
some form of “communication” between the active
sites. In many cases, this is mediated in part by
conformational changes which are transmitted
through the protein subunits. In recent years it has
become increasingly clear that altered protein mo-
tions can also be used to transmit information
through enzymes [45].
Cooperative behaviour by enzymes results in
them not having a rectangular hyperbolic relation-
ship between initial rate and substrate concentra-
tion. A modified form of the Michaelis-Menten
equation, the Hill equation [44] can be used as an
empirical description of their kinetic behaviour:















(22)





In Equation 22, h is the Hill coefficient and K0.5
is analogous to Km in the Michaelis-Menten equa-
tion. However, unlike Km is cannot be represented
as a composite of several discrete rate constants
and should simply be considered as the substrate
concentration required to give an initial rate equal
to half the maximal rate. The Hill coefficient must
be greater than zero and does not have to be an
integer. If it is greater than one, the enzyme is said
to be positively cooperative and if it is less than
one, the enzyme exhibits negative cooperativity. If
Quantitative Enzymology Current Enzyme Inhibition, 2015, Vol. 11, No. 1 27

h=1 then Equation 22 simplifies to Equation 1. In
a positively cooperative enzyme, the Hill coeffi-
cient cannot exceed the number of binding sites
for the substrate in the enzyme; for negatively co-
operative enzymes h cannot be less than the recip-
rocal of the number of binding sites [46].
The dependence of initial rate on substrate con-
centration differs between the two types of coop-
erativity. In positive cooperativity, the graph has a
sigmoidal shape which gets steeper as h increases
(Fig. 11a). This shape of graph clearly illustrates
one of the effects of positive cooperativity which
is to convert enzymes to a more “on/off switch-
like” response. This is especially the case for high-
er values of h where there is a range of substrate
concentrations with negligible activity, a small
transition zone (close to where [S] equals K0.5) and
a region where the rate approaches Vmax (Fig. 11a).
Negative cooperativity gives rise to a graph which,
superficially, looks more similar to the Michaelis-
Menten graph (Fig. 11c). However, the enzyme’s
response to increasing substrate concentration is
more “graded” and a change in substrate concen-
tration will result in a smaller change in rate than
in a non-cooperative enzyme (Fig. 11c). One role
of negatively cooperative enzymes may be to
“buffer” critical systems against changes in sub-
strate concentrations [47].
Since both forms of cooperativity can be de-
scribed by Equation 22, this can be used to deter-
mine the Hill coefficient. Ideally, non-linear curve
fitting should be used; however the presence of
one constant as an exponent can lead to some dif-
ficulties (since small changes in the estimated val-
ue of h can have dramatic consequences for other
values). It can take several rounds of fitting to
produce acceptable results. An alternative method
involves a linearised form of Equation 22:
















(22a)





Using this form of the Hill equation, a graph of
log10 (v/ (Vmax-v) against log10[S] will be a straight
line with gradient equal to h and a y-intercept of –

(a) (b)

(c) (d)

Fig. (11). Cooperativity (a) Positive cooperativity (solid line; data simulated using Equation 22 with K0.5=30 μM, Vmax=100
μMs-1 and h=2) generates a switch-like response when compared to Michaelis-Menten kinetics (dashed line; data simulated
with Km=30 μM and Vmax=100 μMs-1). The two dotted lines from the y-axis indicate 20% and 80% maximal activity. Note how
the corresponding distance on the x-axis (substrate concentration range) is much smaller for the positively cooperative case.
(b) The data from (a) linearised according to Equation 22a. (c) Negative cooperativity (solid line; data simulated using Equa-
tion 21 with K0.5=30 μM, Vmax=100 μMs-1 and h=0.5) generates a graded response when compared to Michaelis-Menten kinet-
ics (dashed line; data simulated with Km=30 μM and Vmax=100 μMs-1). The two dotted lines from the y-axis indicate 20% and
80% maximal activity. Note how the corresponding distance on the x-axis (substrate concentration range) is much larger for
the negatively cooperative case. (d) The data from (c) linearised according to Equation 22a and using similar axes to (b) to
enable comparison.
28 Current Enzyme Inhibition, 2015, Vol. 11, No. 1
log10K0.5 (Fig. 11b,d). One strategy is to use the
linearised form to obtain an estimate of h which is
then used as an initial value in non-linear curve
fitting. Cooperativity can also occur with inhibi-
tors. In such cases a modified form of Equation
22a can be used [48]:

[2]












(22b)





(v0 is the initial rate in the absence of inhibitor and
KI is a constant the exact meaning of which will
vary depending on the type of inhibition present).
CONCLUSIONS AND FINAL COMMENTS
The careful, quantitative analysis of enzyme
catalysed reactions can yield useful information
about the process of catalysis. It is also a vital part
of drug discovery and other processes which re-
quire the identification and characterization of en-
zyme inhibitors. It is important to remember that
enzyme kinetics analysis is a tool to aid under-
standing and that the aim is not solely the collec-
tion of ever-increasing numbers of rate constants.
A dose of realism is often required too. Constraints
(especially limited amounts of enzyme and limited
solubility of substrates) often restrict what can be
done and impose compromises between what
would be ideal from a quantitative enzymology
point of view and what is practically possible. De-
spite these constraints, quantitative enzymology
has been a key part of biochemistry for over 100
years. The robustness and usefulness of these
methods means that they are unlikely to become
obsolete and they will continue to make a contri-
bution for many years to come – especially when
combined with more modern approaches such as
molecular biology and systems biology.
CONFLICT OF INTEREST
The author confirms that this article content has
no conflict of interest.
ACKNOWLEDGEMENTS
My interest in enzymes was largely stimulated
by two excellent lecturers at the University of
Birmingham (UK) – Dr. Eva Hyde and Prof. Chris
Wharton. My continuing interest has resulted from
several research projects and from teaching quanti-
tative enzymology to second year biochemistry
David J. Timson
and medicinal chemistry students at Queen’s Uni-
versity, Belfast (UK).
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Received: March 11, 2015 Revised: May 07, 2015
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Current Enzyme Inhibition, 2015, Vol. 11, No. 1 31
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Accepted: May 08, 2015