In the Classroom
Understanding Enzyme Inhibition
Raymond S. Ochs
Department of Pharmaceutical Sciences, St. John’s University, Jamaica, NY 11439; ochsr@stjohns.edu
Reversible enzyme inhibition is a well-established phenom-
enon; there is little debate about its significance or the math-
ematical equations describing it. However, it is a surprisingly
difficult subject for students to absorb; even established sci-
entists have difficulties with enzyme inhibition. Most come
away from this subject with a good idea of competitive inhi-
bition and a fuzzy notion about everything else! I contend
that the real difficulty is twofold. First, there exists no clear
picture to conceptually illustrate inhibition types other than
competitive. Second, most derivations, particularly at the
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elementary level, focus on the wrong kinetic concepts.
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A Mental Picture
While it is important to formulate a mathematically
unambiguous representation of the ideas of enzyme inhibition
so that they can be put into practical use, the concept that
should come first is often missing. The only currently available
mental picture is that of a “competitive” inhibitor. Here, the
substrate and inhibitor are in competition for the free enzyme.
To arrive at a picture of the other types, we need to consider
mechanistically what the other inhibition forms are.
The basic Michaelis–Menten model describing the
simplest enzyme mechanism is
E+S ES → E + P (1)
The three types of inhibition are competitive, uncompeti-
tive, and mixed.1 For each type, the equation is modified by
the addition of equilibria between the inhibitor, I, and a form
of the enzyme. For a competitive inhibitor, I binds exclu-
sively to E, the free enzyme form, and the extra equation is
E+I EI (2)
Now, if the inhibitor instead binds exclusively to ES, the bound
enzyme form, then the equilibrium that modifies eq 1 is
ES + I ESI (3)
Finally, an inhibitor that has a significant binding to both E
and ES is called “mixed”. In summary,
Inhibition type Equations that describe it
Competitive 1 and 2
Uncompetitive 1 and 3
Mixed 1, 2, and 3
With these basic definitions, we can move to the mental
image. Virtually all students immediately picture competitive
inhibition. Substrates with structural similarity to inhibitors,
but not used in the reaction, are often competitive inhibitors. It
is easy to formulate the idea because we have so many analogies
to draw on. In an ecosystem, different organisms compete
for food; in academic life, students compete for grades; in
financial life, businessmen compete for money.
But what of uncompetitive inhibition? We need an
everyday explanation for the idea that an inhibitor can bind
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
not to the free enzyme, but rather to the ES form. Consider
the analogy of the honey trap. A bear attempts to get honey
from a hive, but owing to a constriction in the top, he can’t
get his paw out after it is filled with honey. He is stuck
(inhibited). Note that the inhibition would not arise unless
he tried for the honey and became “bound” to it. For this
analogy, the bear is the enzyme E; the honey is substrate S;
the hive is the inhibitor I.
Alternatively, consider a law-enforcement “sting” operation.
The suspect is offered an especially attractive deal involving
illegal contraband. When she accepts it, an arrest is made
(she becomes inhibited). Note that inhibition would not
arise unless she tried for the contraband and became “bound”
to it. For this analogy, the suspect is the enzyme E; the
contraband is the substrate S; the law enforcement agent
is the inhibitor I.
Once the picture is grasped, the notion of uncompetitive
inhibitors can immediately become an intuitive one; and since
mixed inhibition merely combines competitive and uncom-
petitive effects, the mental picture for all forms of reversible
inhibition is complete. To extend this understanding as it
directly applies to enzymes, we need to consider the behavior
of the velocity of the enzyme as a function of both changing
substrate and changing inhibitor concentrations. Here too, I
suggest we take an approach that is a departure from tradition
but will lead to a much firmer understanding.
Analysis of Enzyme Velocity Equations
with Reversible Inhibitors Present
Following the lead of virtually all biochemistry texts
and eqs 1, 2, and 3, along with the appropriate conservation
equations for the enzyme forms E, ES, EI, and ESI, we have
three equations for the initial velocity, vi. For competitive in-
hibition,
Vmax S
vi = (5)
I
Km 1 + + S
Ki
for uncompetitive inhibition,
Vmax S
vi = (6)
I
Km + S 1 +
Ki
and for mixed inhibition
Vmax S
vi = (7)
I I
Km 1 + + S 1+
Ki Ki
Note that the last is actually for the case of equal affinities
of I for E and ES; otherwise there would be two different
constants (Kic and Kiu) representing the different affinities.
1453
 In the Classroom
Figure 1. Competitive inhibition. In the direct plot of initial velocity
(vi) vs [S], inhibition is maximal at low [S] (the Vmax/Km tangent
line of the control) and diminishes to zero as [S] approaches infin-
ity (the Vmax asymptote of the control).
Graphs of the results of the three types of inhibition are
displayed in Figures 1–3. I have also plotted two straight lines,
designated as Vmax and Vmax/Km. Vmax is the asymptote of the
control (uninhibited) curve as [S] → ∞. Vmax/Km is the tangent
to the curve at [S] = 0. Clearly, the parameters Vmax and
Vmax /Km characterize the curve; it should also be noted that
they have a simple yet important interpretation in terms of
the mechanism of eq 1. Vmax is the velocity when all the
enzyme is in the form of ES, at high [S]. Vmax/Km is the
apparent first-order rate constant when essentially all the
enzyme is in the form E, at low [S]. These kinetic constants
are therefore our focal points and the key to understanding
enzyme inhibition. Put another way, we are examining the
extremes of substrate effects on velocity in direct space,
rather in reciprocal space (the traditional approach; i.e., the
Lineweaver–Burke plot).
Consider first the competitive inhibition curve (Fig. 1).
At low [S]—near the Vmax/Km line—the competitive inhibitor
causes a substantial drop in enzyme activity; but the inhibitor
has less influence as [S] increases, and in the limit of infinite
[S], there is no inhibition.
In the uncompetitive inhibition curve (Fig. 2), exactly
the opposite behavior is apparent. Here, there is no effect of
inhibitor at low [S]—near the Vmax/K m line—and inhibition
increases as [S] increases, becoming maximally effective as [S]
approaches infinity. While it is true that any ES that is formed
even at low [S] can bind I, the formation of ESI displaces the
equilibrium E + S = ES to the right, just offsetting the inhi-
bition. As [S] becomes saturating, the displacement of the
equilibrium is irrelevant; all the enzyme is in the ES form.
As this is the form to which the uncompetitive inhibitor binds
exclusively, inhibition is maximal.
Finally, the mixed inhibition curve (Fig. 3) is just a
combination of the two we have already considered. This
follows from the fact that the inhibitor binds both the free (E)
and bound (ES) forms of the enzyme. We can appreciate the
result because we already understand these two events in their
extremes—at zero and infinite substrate concentrations. At
low substrate concentration, where the uninhibited velocity
1454 Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
Figure 2. Uncompetitive inhibition. In the direct plot (v i vs [S]) inhi-
bition is maximal at high [S] (the Vmax asymptote of the control)
and diminishes to zero as [S] approaches zero (the Vmax/ Km tan-
gent line of the control).
approaches Vmax/K m, there is no contribution from the un-
competitive inhibitor, so here the mixed inhibitor acts like a
competitive inhibitor; note how the mixed and competitive
inhibition become the same as [S] → 0. At high [S], there is
no contribution from the competitive type of inhibition; here
mixed inhibition approaches uncompetitive. Thus, mixed and
uncompetitive inhibition become the same as [S] → ∞.
Another way of looking at reversible inhibition is that
all three types of inhibition—competitive, uncompetitive, and
mixed with equal affinities for (E) and (ES)—are just limiting
cases of a single more general type in which the inhibitor
has some affinity for both enzyme forms.
Textbook Comparisons
Examination of several current biochemistry textbooks
(1–11) reveals that none have considered enzyme inhibition
from the standpoint of the kinetic terms Vmax and Vmax/Km;
all have used Vmax and Km instead. Virtually all employ double
reciprocal plots as an implicitly essential part of the analysis.
The use of double reciprocal plots for data analysis has
serious disadvantages (12); for pedagogical purposes they
are even more unsuitable, as they confuse more than they
enlighten.
Some treatments (e.g., 13) omit uncompetitive inhibition
because it is rarely encountered. This ad populum argument
is unsound when it is realized that competitive and uncom-
petitive types are extreme forms of inhibition, reflecting the
affinity of an inhibitor for the E or ES form of the enzyme,
respectively. Without understanding each individually, mixed
inhibition has no conceptual basis.
It may also be argued that uncompetitive inhibition is
more generally that form arising when I binds any enzyme
form other than the free form, E. Thus, this inhibition type is
actually common in multisubstrate cases, which are, after all,
also more realistic. Yet introducing multisubstrate kinetics is
an enormous burden on those exposed to kinetics for the first
time. It becomes easier to understand only after the basic idea
of inhibition types is in place.

Figure 3. Mixed inhibition. In the direct plot (vi vs [S]), inhibition is
evident at all concentrations of substrate. Thus the inhibitor behaves
as if it were competitive at low [S] (concentrations where the curve
approaches the Vmax/Km tangent line of the control) and noncom-
petitive at high [S] (concentrations where the control graph ap-
proaches the Vmax asymptote).
Educational Literature
There exists an extensive literature beyond the textbook
treatment, which aims to extend and clarify enzyme kinetics
and enzyme inhibition. The meanings of the kinetic constants
Vmax and Vmax/Km were themselves recently explored (14 ),
although not with an emphasis on enzyme inhibition. In this
article, Northrop re-explains these constants as “release” and
“capture” constants, which is more meaningful than the
standard interpretation, particularly with more complex enzyme
mechanisms than the one examined here. This treatment also
shows the value of reciprocal plots for a deeper understanding
of these constants. However, the approach is far more advanced
and general than the one presented here and befits the advanced
student rather than the beginner who needs a fundamental
understanding of enzyme inhibition.
Another approach is the presentation of computer programs
to allow direct visualization of kinetic results (15, 16 ). Still
others emphasize various types of graphical transformations that
Table 1. Influence of an Enzyme
Inhibitor on Kinetic Constant Pairs Vmax,
Km and Vmax, Vmax /Km
Effect of Inhibitor on
Inhibition Type
Vmax Km Vmax Vmax /Km
Competitive — —
Uncompetitive — Scrimgeour, K. G. Principles of Biochemistry; Prentice Hall:
Mixed — Englewood Cliffs, NJ, 1996.
N OTE: When enzyme inhibition is described in terms of the pair
Vmax, Km, the changes are confusing. The common interpretation of Km as
an affinity constant, in which changes are inverse to the expected rate,
incurs the double problem of dealing with inverses and having an
inhibitor exert an apparent stimulation (uncompetitive case). Moreover,
the fact that Km is unchanged in the mixed case leads to no mechanistic
insight. The opposing symmetry between competitive and uncompetitive is
not apparent at all using the Vmax, Km pair. With the pair Vmax, Vmax/Km,
all interpretations are direct, no assumption of affinity is needed, and
symmetry is obvious.
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
In the Classroom
may allow more comprehensive analysis (17), although the
aim here is really to extract constants rather than to explain
fundamentals. Finally, there are extensions of enzyme kinetics
to enzymes acting at membrane surfaces, where the kinetics
are dominated by the surface interactions and become quite
different (18). None of these approaches specifically addresses
a new way to fundamentally view enzyme inhibition.
The Advantages of Vmax/Km
Table 1, modified from Cornish-Bowden (19), compares
the use of the pair Vmax, Km with the pair Vmax, Vmax/Km in
the analysis of reversible inhibitors. Note how the symmetry
in the inhibition types emerges readily when Vmax/Km and
Vmax are taken as kinetic constants. Interpretation of events
using the pair Vmax and Km is far more complicated.
A good deal of confusion arises from the more traditional
focus on Km instead of Vmax/Km. Thus, Nahorski et al. (20)
described uncompetitive inhibition as “unusual and counter-
intuitive”. Fell (21) similarly described uncompetitive inhibition
as having “strange properties”, with “contradictory actions …
reducing the apparent limiting rate (like a noncompetitive
inhibitor), but at the same time it reduces the Km of the
enzyme for its substrate, which would normally cause an
activation”. This confusion of an inhibitor causing an “activa-
tion” stems entirely from trying to use Km rather than Vmax/Km
to describe it.
I realize it will be difficult for the more traditionally
oriented to use direct velocity–substrate plots and embrace
Vmax/Km for the understanding of enzyme inhibition. More-
over, many are more interested in discussing more elaborate
mechanisms, of disdaining the simple Michaelis–Menten
kinetic ideas in favor of more complex notions. However, all
the more complex notions are, in one way or another, based
on this bedrock. If it is not fully and clearly understood, our
grasp of the more esoteric notions may seem lofty, but have
no foundation.
Note
1. There are also problems in nomenclature. Mixed inhibition
is also called noncompetitive, which leads to some conflict with un-
competitive. Some differentiate mixed from noncompetitive. Since our
objective is the most effective teaching of the concepts, the word
mixed is used throughout this treatment. After the idea is clear, it
is a simple matter for students to learn alternative definitions and
more formal treatments.
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