Addison Aulf

Cornell College
Mt. Vernon, Iowa 52314

An Introduction to Enzyme Kinetics

The purpose of this paper is to present a simple, intuitive, yet accurate introduction to some of the fundamental
concepts of enzyme kinetics. The experimental definitions
of competitive, uncompetitive, and noncompetitive inhibition will be given, and a simple mechanistic model will
be presented for each case. While these models may be
too simple to adequately represent most actual enzymatic
reactions, they correctly indicate possible modes of action
of enzyme inhibitors.

late

Characteristics of Enzyme Kinetics

Most organic reactions show a linear dependence of the

rate upon the concentrations of the reactants. For example, the kinetics of many nucleophilic substitution reactions show that the rate is directly proportional to both
the concentration of the nucleophile and the concentration of the substance undergoing substitution ( S d kinetics). In other reactions the rate of the reaction may be independent of the concentration of one of the reactants,
and many other nucleophilic substitution reactions show
this type of behavior (SN1kinetics). In contrast, however,
to most organic reactions, enzyme catalyzed reactions
usually show a nonlinear dependence of the initial rate of
reaction on the concentration of a reactant, or substrate,
S . This characteristic variation of initial rate with substrate concentration, [S], is shown in Figure la. The equation for a line of this form, a hyperbola, is given by

1
rate

(1)

Thus, as in the case of a straight line, two parameters determine the line, and a plot of experimentally determined
values of the initial rate of an enzymatic reaction versus
[S] will yield information only as to the values for the two
parameters.
The physical significance of the parameter a can be
seen by considering eqn. (1) a t high S concentrations. At
high [S], eqn. (1) reduces to: rate = a, since the value of the
fraction in the parenthesis approaches one as [S] increases.
The parameter a is thus seen to be the maximum initial rate
of reaction which can he attained by having S present a t
high concentration. The value of a can be estimated from
the intercept of the upper horizontal dotted line of Figure 2.
The physical significance of the parameter b can be understood by considering eqn. (1) when b and [S] are equal.
When this condition is met, eqn. (1) reduces to: rate =
a(1/2). Thus b equals that concentration of S a t which the
rate is half its maximum possible value. The smaller b, the
more rapidly the rate of the reaction will rise toward a with
increasing [S]; b is a measure of the sensitivity of the rate to
changes in [S]. The value of b can be estimated from the
intercept of the vertical dotted line of Figure 2.

Figure 1. a, Characteristic variation of initial rate with substrate concentration. No inhibition. b, Double reciprocal plat of la.

shown by Lineweaver and Burk, taking the reciprocal of

both sides of eqn. (1) gives
b 1
rate
Thus one can see that a plot of (llrate) versus (l/[S])
will give a straight line whose intercept on the (ljmte)
axis will be l/a, and whose slope will be bla. The value of
a is then the reciprocal of the intercept on the ordinate,
and the value of b is the value of the slope times a. Figure
l b shows the double reciprocal plot which corresponds to

Lineweaver-Burk or Double Reciprocal Plots

At this point, we will consider an alternative method of

obtaining values of the parameters a and b fmm values of
initial rate of reaction a t different concentrations of S . As

[SI
Figure 2. Determination of thevalue of parameter a.

Volurne51, Nurnber6. June 1974 / 381

Fieure l a . The advantaee of analvzine

the data in terms
of;
"double reciprocal" plot is that
it is easier to accuratelv fit and extra~olatea straight
- line
than a curved line.
So far, we have not said a word about a possible enzymatic mechanism, or model, which could account for the
kmd of kinetic behavior summarized in eqn. (1) and represented in Figure 1. Indeed, to do so would have been
putting the cart before the horse, or the interpretation hefore the experiment.

fi in ewe aver-^+'&

A Model Wilhout lnhibition

A particularly simple model for an enzymatic reaction

whose kinetics can be expressed by eqn. (1) is
E

S-P

If the reaction were ta occur according to the scheme

be present as E, and the rate therefore will he half maximal. In addition, if b is large, it means that either k-I, or
k,, or both are large relative to kl (it means that ES
tends to break down more rapidly to give E than it is
formed from E) and so it will take a higher concentration
of S to provide a particular steady-state concentration of
ES. In other words, the larger b, the more slowly the rate
will increase with increasing [S].
In most discussions of enzyme kinetics, the expressions
for a and b which are derived on the basis of this mechanism are given special names: kp[Eo] is called V,.,, and
(k_l k.,)/k~ is called KM, the Michaelis constant. It is
important to realize that KM does not generally equal
k - ~ / k ~or, KI, the equilibrium dissociation constant for
the combination of E with S. KM and KI will he equal if
and only if k, <j: k-I, and to simply assume that KM
equals K1 is always a guess and is often a mistake.
The values for a and b which were deduced from this
model by inspection of eqn. (4) could also have been obtained by taking the reciprocal of both sides of eqn. (4)

h-,

which represents the steps

+ k,

1 =--

k,[EJ

rate

ES * P + E

the following kinetic behavior can be deduced. The rate of

the overall reaction is the rate of the second step, the decomposition of an enzyme-substrate complex to give the
product, P, plus enzyme: rate = kp[ES]. Since the concentration of the complex, [ES], cannot he measured directly, one must express @S] as the known total enzyme
concentration, [Eo], times the fraction of the enzyme present as ES. In this case, the total enzyme concentration,
[Eo], equals the sum of the concentrations of the free enzyme, [El, and enzyme with bound substrate, [ES]

This shows that a plot of (l/rate) versus (l/[S]) would

give
hi

intercept = -- = h,,[&,l a

+ h.

k
b
and slope = A = kdEJ

and that
a = kJEJ =

Vmax

Inhibition

The ratio of free enzyme to bound enzyme, [E]/[ES], is

calculated by assuming that shortly after the reaction hegins, the E S concentration has risen to and is maintained
at a constant value. During this steady state, which exists
throughout most of the time of the reaction, the rate of
formation of ES equals its rates of reaction

and therefore

Substituting this into eqn. (3) one obtains

an equation of the same form as (I), which indicates that

according to this mechanism
a = h,[E,I

k-1 + h,
and b = h,

This result makes sense for a, the maximum possible initial rate of reaction, because the rate is always kp[ES] and
the maximum possible value for [ES] must obtain when
"all" the enzyme is present as ES; that is, when [ES] =
[Eo]. This result also makes sense for b, that value of [S]
for which the rate is half its maximum value, since when
kp)/kl equals [S], the ratio [E]/[ES] is one. At
(k_l
this [S], half the enzyme will be present as ES, half will

382

Journal ot Chemical Education

Certain substances reduce the rates of enzymatic reactions. Such substances are called inhibitors. In terms of
experimentally obsewahle effects, two types of inhibition
can he distinguished. The first is characterized by an increase in the slope of the Lineweaver-Burk plot when I is
present, hut an unchanged intercept. This type of inhibition is called competitive inhibition. In the second type of
inhibition, the effect of the inhibitor is the opposite: in
the presence of the inhibitor, the slope of the LineweaverBurk plot is unchanged, hut the intercept is increased.
This type of inhibition is called uncompetitiue inhibition.
These two modes of inhibition can also he described in
terms of the graph of rate versus [S]. In competitive inhibition, the rate of reaction rises more slowly in the presence of the inhibitor, but finally approaches the same
maximum rate which is attainable in the absence of the
inhibitor. In uncompetitive inhibition, the initial rate of
reaction rises with increasing [S] as quickly as in the absence of the inhibitor,' hut only to approach a lower maximum rate of reaction than that attainable in the absence
of inhibitor. In comnetitive inhibition. the further addition of S can restore the rate to what it'was in the absence
of I. hut with uncom~etitiveinhibition. this does not ha^Figures 3 and 4.illustrate competitive and uucompetitive inhibition in terms of plots of rate versus IS1,
. .. and of
(I/rate) versus (l/[S]).
When both of these inhibition effects are observed together, noncompetitiue inhibition is said to be involved.
Figure 5 illustrates noncompetitive inhibition in terms of
plots of rate versus [S] and (l/rate) versus (l/[S]).
'The initial slope. alb, is the same.

COHPETITIYE INHIBITION

&l

Figure 3. a, plot ot rate versus substrate concentration. Competitive inhibition, b. Double reciprocal plot of 3a.

I t is important to realize that inhibition bas been defined without reference to any mechanistic model for inhibitor action. In the next three sections, we will consider
simple models for competitive inhibition, uncompetitive
inhibition, and noncompetitive inhibition.

rate versus substrata concentration. uncompetitive

Figure 4, a, Plot
inhibition. b, Double reciprocal plot of 4a.

EUNCOYPETITIY~

~NH~B~TION

A Model for Competitive Inhibition

A possible model for the reaction

S

for which I is a competitive inhibitor is the following

scheme
E

This scheme represents the steps

,k

and from these reactions the following kinetic behavior

can be derived. The rate of the overall reaction is the rate
of decomposition of E S to give product: rate = k,[ES].
Fieplacing [ES] by [Eo] times that fraction of the enzyme
which is present as ES one obtains
rate

k 6 ~ a 1([ES]

SI
+P [El
+ PE])

d7
b

(5)

Fiaure 5. a. Plot of rate versus substrate concentration. Noncompetitive

inhibition. b. Double reciprocal plot of 5a.

Volume 5 1 , Number 6. June 1974

383

Again, assuming a steady-state concentration of ES, one

can equate the rates of formation and use of ES to find that

This scheme represents the following steps

Assuming also a steady-state concentration of IE, one can

write
and see that

UEI MI1 - PI
K,
[El - k-,
(Kz is the dissociation constant for the reaction IE e I +
E). Realizing that [IE]/[ES] = ([E]/[ESl) ([IE]/[E]), one
substitutes into eqn. (5)and obtains

a rate expression which can account for the characteristics

of competitive inhibition. When [S] is large, the fraction
in the brackets will approach one and the rate will hecome equal to k,[Eo] or VmaX.When [I] is large or IE is
not easily dissociated (that is, Kz is small), inhibition will
be great because of a high ratio of IE to ES.
When the reciprocal of each side of eqn. ( 6 ) is taken,
one obtains

ha
*
IES
k-,

E+I

k B I I ] = kLJIE]

The rate of the overall reaction is the rate of decomposition of ES to give product: rate = k,[ES]. Again, expressing [ES] as [Eo] times that fraction of E o present as
ES gives
[ESI
k"Eol ([ES] +[El + UES]) =

Assuming a steady-state concentration of ES, one can

write

k,[EKSl + h,[IESl=

kdESl+ k-,[ES]+ h,[ESKIj

If IES is also present a t a steady-state concentration

k-LIES] kJESII]

and it then follows both that

[El
K,w
[IES]
[ES] - [S] and [ES]

h [l] = [I1
I

(Ks is the dissociation constant for the reaction IES r ES

I). Substituting into eqn. (7) one obtains

+
from which it is clear that a plot of (l/rate) versus (l/[S])
will give
1

intercept = -I-= -a and slope =

kdEJ

kLEo1

=-

and therefore
a = kdEJ =

Vm.,

and

From this rate expression, it is apparent that no matter

how great [S], the fraction in the parenthesis will never
approach one for finite P] and K3. This accounts for the
observation that increasing [S] cannot overcome the effect
of I in uncompetitive inhibition.
Taking the reciprocal of both sides of eqn. (8) gives

[I1

I+1 =-rate
kp[2

Here we see that this kinetic scheme predicts that the

value of the intercept of the Lineweaver-Burk plot will be
the same as in the ahsence of the inhibitor, but that the
slope will he (1 ([I]/Kz)) times greater than in the absence of the inhibitor.
It should also be clear that Kz, the dissociation constant
for IE, can be calculated from either the new slope or
from b, knowing [I]and KM.
From the expression for b in the presence of inhibitor, it
is apparent that the greater [I] or the smaller Kz, the
more difficult it will be to overcome the effect of I by addition of more S since IS] must equal b to attain half the
maximum rate.

+[(&)

&I

from which it can be seen that a plot of (l/rate) versus

(l/[S])will give

Therefore

A Model for Uncompetitive Inhibition

For the reaction

for which I is an uncompetitive inhibitor, the following

scheme is possible

384

Journal of Chemical Education

This mechanism accounts for the increased intercept and

unchanged slope of the Lineweaver-Burk plot which are
characteristic of uncompetitive inhibition. In addition, it
can be seen from the expression for a that the maximum
possible rate in the presence of inhibitor will be less than
in its absence, which accounts for another of the defining
characteristics of uncompetitive inhibition.
From the expression for b, it appears that a smaller
concentration of S will cause the reaction to proceed a t
half its maximum possible rate in the absence of inhibi-

tor. For example, when p] = Ka,half the maximum rate

possible in the presence of this level of inhibitor is Vmor/
4, and the concentration of S required to achieve this rate
is K M / ~Otherwise,
.
when no inhibitor is present, half the
maximum rate is Vm,,/2, and the concentration of S
needed to achieve this rate is KM. In both these cases,
half the enzyme molecules, on the average, have an S
molecule bound to them: in the first case as equal
amounts of ES and IES (Y4 of the enzyme molecules are
' as E); and in the second
present as ES, Y4 as IES, and k
as ES only ('k of the enzyme molecules are present as E S
and 'h as E). It appears that in the presence of inhibitor,
S can be more effectively bound to the enzyme a t a given
concentration of substrate. This surprising result can be
understood in the following way. The effect of the inhihitor a t a concentration equal to K3 is to take half of the
enzyme molecules present as E S and to nail the S molecule to them to give IES. Since the concentration of S is
always far greater than that of E, this will reduce the concentration of free S by an insignificant amount but will
also make it necessary that only
of the enzyme molecules be present as ES to achieve the maximum possible
rate at this level of I. Thus the [S] required for half the
maximum rate (that is, the value of b) is reduced hy a
factor of two when p] = K3. I t is as if, when V] = K3, one
is working with only half as much enzyme since a t any
time half the enzyme with S bound to it is present as the
unreactive IES.

from which one can show that a plot of (l/rate) versus

(l/[S]) will give

and that

This model, which includes the possibility of forming

both IE and IES, predicts that both the slope and the intercept of the Lineweaver-Burk plot will he greater in the
presence of I than in its absence, and thus accounts for
the experimental characteristics of noncompetitive inhihition. The expressions for a and b indicate that in noncompetitive inhibition, the maximum reaction rate, a, will always be less than Vmal, but that b may he greater or less
than KM, depending, in this model, upon whether Ka is
smaller than Ks, or vice versa.
A
- - little studv of this model will reveal that if Ks is very
large (IES d&s not form) it reduces to the competitive
model: if K9 is verv laree (IE does not form) i t reduces to
the uncomp&itive model; and if both Kz and K Z are very
large, I does not act as an inhibitor. The noncompetitive
model includes the other three as special cases.
It is interesting to notice that a variation of this model
could include a direct interconversion of I E and IES in
addition to the other reactions. The equilibrium constant
corresponding to this interconversion would have to equal
K I K ~ / Ksince
~ the relative amounts of I E and IES are established as soon as those three equilibrium constants are
specified. These two versions of this model cannot be distinguished hy the experiments described.
~~

A Model for Noncompetitive Inhibition

For the reaction

E

SAP

for which I acts as a noncompetitive inhibitor, the following scheme is possible

This scheme represents the following steps

For this mechanism, the rate of the overall reaction is the

rate of conversion of ES to E and P: rate = k,[ES]. Expressing [ES] as [Eo] times that fraction of Eo present as
ES gives

Again, assuming steady-state concentrations of ES, IE,

and IES, and proceeding as before, one can write

Summary
The table summarizes the expressions derived for l / a
and b/n (the intercept and slope of the Lineweaver-Burk
plot) and a and b for four experimentally distinguishable
mechanisms for the enzyme catalysed conversion of S to
P. The four mechanisms account for no inhibition, competitive inhibition, uncompetitive inhibition, and simultaneous competitive and uncompetitive, or noncompetitive, inhibition.
As illustrated by the model, competitive inhibition is
usually explained by a mechanism in which I and S combine with the same enzyme species (in this case they compete for E ) and the binding of either I or S by that enzyme species renders it unable to hind the other (neither
IE nor E S can form IES).= In this type of mechanism, a
sufficiently high concentration of S can keep I from binding and eliminate its effect.
Also, as illustrated by the model, uncompetitive inhibition is usually explained by a mechanism in which S and
I combine with different enzyme species. In the model, S
combines with E but not with ES, while I combines only
with ES and not with E. The uncompetitive inhibitor
thus reduces the concentration of an enzyme species in
=An exception may occur when two enzyme forms are in rapid
equilibrium with one another so that depletion of one form will
lead to simultaneous depletion of the other. In such a case, S
could react with one form and I with the other, and the kinetics
of competitive inhibition would be observed. The two farms might
be conformationalisomers.
Volume 51. Number 6. June

1974

385

Summary of Expressions for Various Mechanisms for the Enzyme Catalyzed Conversion of S to P

1
a

Type of Inhibition

b
a

none

uncompetitive

such a way t h a t increasing t h e concentration of S cannot

overcome t h e effect.
If the inhibitor can participate i n both ways, simultaneous competitive and uncompetitive inhibition take
place, and noncompetitive inhibition is said t o occur.
T h e experimental criteria and mechanistic interpretation of t h e three types of inhibition may b e summarized
in this way.
Competitive Inhibition
Experimental: 1) Intercept of Lineweaver-Burk plot unchanced.
~
~
~
~
~
2) Slope of Lineweaver-Burk plot increased.
3) Effect of I can he overcome by addition of S;
(maximum initial rate = a = Vmoz).
4) Apparent Michaelis constant = b > K M
Interpretation: Presence of I reduces the concentration of the
enzyme species which can bind S.
n~~~~

not bind free S. This lowers the concentration

of the enzyme in the various species present when
I is absent, and does it in such a way that the
addition of S cannot overcome the effect.
NoncompetitiveInhibition
Experimental: 1) Intercept of Lineweaver-Burk plot increased.
2) Slope of Lineweaver-Burk plot increased.
3) Effect of I cannot be overcome by addition of
S; (maximum initial rate = a < Vmaz).
4) Apparent Michaelis constant may he larger
than, smaller than, or equal toKnr.
Interpretation: Joint occurrence of the two actions described
for competitive and uncompetitive inhibition.

Literature
some of the iiteretur. which I h n d most "Saf"1 and interrating includes the fol-

Uneompetitive Inhibition
Experimental: 1) Intercept of Lineweaver-Burk plot increased.
2) Slope of Lineweaver-Burk plot unchanged.
3) Effect of I cannot be overcome by addition of
S; (maximum initial rate = a < Vmax).
4) Apparent Michaelis constant = b < KM
Interpretation: I combines with an enzyme species which does

386 / Journal of Chemical Education

lwhg:

A. G..Dlsrvasions Pornday Soc., No. 20. p. 161 (1955).(Activation and InhihitionofE"zyrnes1.

Alherty, R.A..Adoon. inENymol., I?. l(1956J.(EnwmeKinetiea).
King. E. L., and Altman, C., J Phys. Chem., 60. 1375 (19561.(A Sehemstie Method of
~ e t i v i n g t h e R a Lt ~a w s fmEnryme-Catalysed ReastionsJ.
Omton,

Cldand, W. W., "The Enzymn," (Editor Boyc., P. D.), 3rd Ed., Academic P.es,
NealYork, 1910, pp. 1-65. (Steady State Kinetics).
Plowman. Kent, "Enzyme Kinetic$/ MeCraw-Hili Bmk Co., New Ymk, 1913.