Michaelis-Menten Kinetics☆

Robert Roskoski, Blue Ridge Institute for Medical Research, Horse Shoe, NC, USA
ã 2015 Elsevier Inc. All rights reserved.

Saturation Kinetics 1
The Specificity Constant 2
Derivation of the Michaelis-Menten Equation: Briggs-Haldane Treatment 2
Linear Plots 4
Multisubstrate Reactions 5
Steady-State Rate Equations for Enzymes with Two Substrates and Two Products 6
Determination of the Kinetic Constants for Enzyme-catalyzed Reactions 7
Enzyme Inhibitors 7
References 9

In the study and characterization of an enzyme, it is important to know the optimal concentration of its substrates. These
concentrations are related to the Km value, which is the substrate concentration that results in a reaction velocity that is one-half
of the maximum or Vmax/2. Determination of these values is important in developing and using enzyme assays for studying normal
physiological processes and for defining drug interactions with an enzyme.

Saturation Kinetics

The study of reaction rates is the science of kinetics. For many chemical reactions, the rate is proportional to the concentration of a
reactant [A] as expressed by the following:

Velocity ¼ K1 ½A1 ¼ K1 ½A  [1]

Velocity ¼ K0 ½A 0 ¼ K0 [2]
where k1 is the first-order rate constant and [A] is the concentration of A. If the velocity is independent of the concentration of A, the
exponent is zero and the reaction is described as zero-order with a rate equal to k0. Note that the order of a reaction with regard to a
specific reactant is related to the exponent of that reactant. In the first equation, the power of [A] is 1, and the reaction is first order
with respect to [A], meaning that the reaction rate is directly proportional to [A]. In the second equation, the power of [A] is zero,
and the reaction is zero order with respect to [A]. This condition is met when an enzyme becomes saturated with substrate (i.e., the
rate is independent of [A]) because the reaction velocity will not increase appreciably by increasing the concentration of substrate.
Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions. Typically a plot of the initial velocity of an enzyme as a
function of substrate concentration yields a rectangular hyperbola (Figure 1). As the substrate concentration is increased, the
increase in activity becomes progressively smaller and approaches saturation. In an uncatalyzed process, the reaction increases
indefinitely as reactant concentration increased. At low concentrations, an increase in the substrate concentration leads to a linear,
or first-order, increase in the rate of an enzyme-catalyzed reaction. At very high concentrations, an increase in substrate concen-
tration leads to a very small increase in the rate of an enzyme-catalyzed reaction with zero-order kinetics (the velocity appears to be
independent of substrate concentration).
The Michaelis-Menten Equation. Consider the enzyme-catalyzed conversion of substrate (A) to product (P).

Michaelis and Menten assumed that the enzyme and substrate are at equilibrium during catalysis and that the rate of product
formation does not perturb this equilibrium. The equilibrium is expressed by the dissociation constant for the EA complex.

Ks ¼ ½E½A =½EA [4]

The contemporary Michaelis-Menten equation relates the velocity of an enzyme-catalyzed reaction to two kinetic constants
(Vmax and Km) where Km is related to Ks as noted later. This equation is expressed as follows:

v ¼ Vmax ½A =ðKm + ½A Þ [5]

☆
Change History: August 2014. R Roskoski New abstract, key words, all new figures, new section on enzyme inhibitors.

Reference Module in Biomedical Research http://dx.doi.org/10.1016/B978-0-12-801238-3.05143-6 1

2 Michaelis-Menten Kinetics

Figure 1 The rate, or velocity, of an enzyme-catalyzed reaction as a function of substrate concentration. The curve is a rectangular hyperbola. The Km is
the substrate concentration at half-maximal velocity, which corresponds to Vmax/2.

where v represents the reaction velocity, Vmax is the maximal velocity, Km is the substrate concentration at half-maximal velocity,
and [A] is the substrate concentration. Under defined conditions and specific amounts of enzyme, an enzyme exhibits a maximum
velocity (Vmax), which is approached as a limiting value as the substrate concentration increases. This equation was derived in 1913
by Leonor Michaelis and Maude L. Menten, a post-doctoral associate. Both of these investigators were trained as physicians and
biochemists.
The Michaelis-Menten equation is valuable because it provides a tool for understanding enzymatic reactions. When v ¼ Vmax/2,
Km ¼ [A]. That is, the Km is the substrate concentration at Vmax/2. It is instructive to use the Michaelis-Menten equation to calculate
some reaction velocities under the following conditions. Set the Vmax at 100 mM min1 and the Km at 1 mM and then calculate the
velocity (v) at the following substrate concentrations: 0.1 mM, 1 mM, 10 mM, 100 mM, and 1000 mM. Note there is a large
increase in reaction velocity in going from 0.1 mM to 1 mM, and there is a small increase in velocity in going from 100 mM to
1000 mM. The Km is a constant, and it is a concentration. Moreover, the Km is a constant derived from rate constants (as shown
later). The Vmax, the theoretical maximal rate of the reaction, is also a constant. To reach Vmax requires that all enzyme molecules are
bound with substrate. The Vmax is asymptotically approached as substrate is increased (Figure 1). When [A] is low, the rate equation
is first order in [A]. When [A] is high, the equation for rate is zero order in [A]. The Michaelis-Menten equation describes a
rectangular hyperbolic dependence of v on [A]. An initial characterization of an enzyme generally involves the determination of the
Km, or Michaelis constant.

The Specificity Constant

The turnover number of an enzyme (kcat or catalytic rate constant) is the maximal number of molecules of substrate converted to
product per active site per unit time of several different substrates to different products. The kcat/Km value, or specificity constant, of
the various substrates can be compared. That substrate with the highest value is the best substrate for the enzyme, accounting for the
name specificity constant. The rate of any reaction is limited by the rate at which reactant molecules collide. The diffusional limiting
rate for a bimolecular reaction is 108 to 109 M1 s1. The ratio of kcat/Km is a first-order rate constant. The product of kcat/Km and the
substrate concentration (at subsaturating levels) yields the rate of the enzyme-catalyzed reaction. This rate is proportional to the
substrate concentration and is therefore designated first order. Enzymes that have ratios of kcat/Km near 108 to 109 M1 s1 (close to
the maximum allowed by the rate of diffusion) have achieved catalytic perfection. Triose phosphate isomerase (EC 5.3.1.1, its
enzyme commission number), an enzyme of the glycolytic pathway, is an enzyme that has this attribute. Most enzymes, however,
have specificity constants orders of magnitude below this value.

Derivation of the Michaelis-Menten Equation: Briggs-Haldane Treatment

The conversion of substrate A to product P with the enzyme E may be illustrated by the following equation:
 Michaelis-Menten Kinetics 3

The rate constants for the reactions are indicated by k. Consider only the initial reaction rate when the concentration of product
is zero and assume that the concentration of substrate exceeds the concentration of enzyme by several orders of magnitude (the
usual situation used during enzyme activity measurements).
Briggs and Haldane first formulated the steady-state analysis, which states that the concentration of the enzyme-substrate
complex is constant during enzyme catalysis (Segel, 1975).

d½EA=dt ¼ 0 [7]

This differential equation indicates that the enzyme-substrate concentration is constant during the assay, a condition that
usually occurs within milliseconds after the initiation of the enzyme activity measurement. Next,

Rate of EA formation ¼ K1 ½Ef ½A  + K2 ½Ef ½P ; [8]

where [Ef] is the concentration of the free enzyme. The second term in this equation can be deleted because [P] is essentially zero in
the early part of the initial velocity measurement and the rate of EA formation from P is negligible. Because the concentration of the
substrate A is much greater that the concentration of the enzyme, [A] is constant. The above equation reduces to the following:

Rate of EA formation ¼ K1 ½Ef ½A  [9]

Proceeding in a similar manner:

Rate of EA breakdown ¼ K1 ½EA + K2 ½EA  ¼ ðK1 + K2 Þ½EA  [10]

Substituting eqns [9] and [10] into eqn [7] yields the following steady-state expression:

d½EA=dt ¼ K1 ½Ef ½A   K1 ½EA  K2 ½EA  ¼ 0 [11]

During the steady state, the rate of formation of the complex equals the rate of breakdown as shown in eqn [12]:

K1 ½Ef ½A  ¼ K1 ½EA + K2 ½EA [12]

Because there are two forms of the enzyme – free enzyme [Ef] and substrate-bound enzyme [EA] – the total enzyme
concentration [E] can be expressed as being equal to the sum of the two terms:

½E ¼ ½Ef  + ½EA

or

½Ef  ¼ ½E  ½EA  [13]

Substituting the right-hand portion of eqn [13] into eqn [12] yields:

k1 ð½E  ½EAÞ½A  ¼ ðk1 + k2 Þ½EA

or

ð½E  ½EA Þ½A  ¼ fðk1 + k2 Þ=k1 g  ½EA  [14]

Define a new constant, Km, such that

Km ¼ ðk1 + k2 Þ=k1 [15]

The constant, Km, is called the Michaelis constant. Substituting this into eqn [14] yields

ð½E  ½EAÞ½A  ¼ Km ½EA

or

½E½A   ½EA ½A  ¼ Km ½EA  [16]

Michaelis and Menten proposed that the reaction rate is proportional to the concentration of the EA complex, a key concept.
Therefore,

v ¼ k2 ½EA  [17]

Rearranging
4 Michaelis-Menten Kinetics

½EA  ¼ v=k2 [18]

Substituting v/k2 for [EA] in eqn [16] leads to the following rate equation:

½E½A   v=k2  ½A  ¼ Km  v=k2 [19]

Multiplying each term of eqn [19] by k2 yields:

k2 ½E½A   v½A  ¼ Km v [20]

Another key concept developed by Michaelis and Menten is that the maximal velocity represents the condition when all enzyme
is converted into EA, the enzyme-substrate complex. Under this condition, Ef is equal to zero, and [E] equals [EA]. To obtain the
maximal velocity, replace [EA] with [E] in eqn [17]:

Vmax ¼ k2 ½E [21]

Inserting eqn [21] into the left-hand side of eqn [20] yields

Vmax ½A   v½A  ¼ Km v [22]

Finally, rearrangement yields the Michaelis-Menten Equation

v ¼ Vmax ½A =ðKm + ½A Þ [23]

The original formulation of the Michaelis and Menten equation differed somewhat from that given above. Michaelis and
Menten assumed that k2 is  k1 and k1; they defined a constant Ks ¼ k1/k1 (which is the dissociation constant of the EA complex
into E + A). The equation they derived is nearly the same as eqn [23], namely, v ¼ Vmax [A]/(Ks + [A]). The Michaelis constant (Km) is
generally not equal to the dissociation constant (Ks) of an enzyme for the substrate. The dissociation constant is the concentration
of substrate when half the enzyme is free and half is occupied with substrate under nonturnover conditions. The Km and Ks
represent distinct entities and the Km is the more general case. When k2 is  k1 and k-1, then k2 can be ignored in the equation that
defines the Km (Km ¼ (k1+ k2)/k1). Thus, when k2 is small, then Km  k1/k1  Ks.
The Michaelis constant is a property of every enzyme and is usually near the physiological concentration of the substrate. The
Michaelis constant is made up of rate constants for the formation of the enzyme-substrate complex and the decomposition of the
complex to form enzyme and substrate or product. For multisubstrate reactions, the Michaelis constant is the ratio of numerous
kinetic constants (Cook and Cleland, 2007).
A comparison of the Michaelis constants for hexokinase (EC 2.7.1.1) and glucokinase (EC 2.7.1.2) for glucose is informative.
Hexokinase is found in all human cells, including liver, and has a Michaelis constant for glucose of about 30 mM. Glucokinase is
found in liver, and it has a Michaelis constant for glucose of 10 mM. At the postprandial concentrations of glucose in the hepatic
portal vein (several millimolar), hexokinase is operating at its maximal velocity. In contrast, glucokinase is not saturated and can
metabolize more glucose as the substrate concentration increases.

Linear Plots

Because it is difficult to accurately determine the Vmax for an enzyme by examining a rectangular hyperbola, it is also difficult to
establish the value of the Km in this way. However, it is informative to plot the data in a linear form to determine whether an
enzyme follows Michaelis-Menten kinetics or whether enzyme behavior deviates from this. Moreover, such plots may also indicate
data points that are ‘outliers.’ The Lineweaver-Burk plot is the most commonly used linear plot for enzyme kinetic analysis
(Figure 2). Today enzyme kinetic constants are determined by fitting experimental data to rate equations by computer. Moreover,
Cleland has argued that enzyme kinetic data should not be analyzed by linearization methods such as the Lineweaver-Burk plot,

Figure 2 Lineweaver-Burk plot.

 Michaelis-Menten Kinetics 5

but rather by fitting the non-transformed kinetic data to rate equations by non-linear least squares analysis (Cleland, 1963a).
Lineweaver-Burk plots are currently used for illustrative purposes based on kinetic constants determined by computer algorithms.
The Lineweaver-Burk equation represents the reciprocal of the Michaelis-Menten equation:

1=v ¼ ðKm + ½A Þ=Vmax ½A Þ ¼ Km =ðVmax ½A Þ + ½A Þ=Vmax ½A Þ [24]

1=v ¼ Km =Vmax  1=½A  + 1=Vmax [25]

This equation can be compared with the equation for a straight line: y ¼ mx + b, where m is the slope and b is the y-intercept. In
the Lineweaver-Burk equation, Km/Vmax is the slope (or m) and 1/Vmax is the y-intercept (or b). For enzymes that obey Michaelis-
Menten kinetics, when the reciprocal of the substrate concentration is plotted versus the reciprocal of the velocity (1/v), results
similar to those displayed in Figure 2 are obtained.
The value of 1/Vmax is obtained by extrapolation to the y-axis, and the maximal velocity corresponds to the velocity at an infinite
substrate concentration (1/[A] ¼ 0). The plot also yields 1/Km. Because it is a reciprocal plot, a larger Vmax corresponds to a smaller
value of the ordinate (y-axis). Similarly, a larger Km corresponds to a less negative value of the abscissa (along the x-axis). The
x-intercept and 1/Km are negative, and Km is positive (a substrate concentration cannot be negative).

Multisubstrate Reactions

Enzyme reactions with a single substrate and a single product, such as those described above, are called uni-reactions and represent
a minority of the reactions that occur in metabolism. For multisubstrate reactions, reactants are designated A, B, C, D. . . for the uni-,
bi-, ter-, and quad-reactant substrates. The products are designated P, Q, R, S. . .. The Michaelis-Menten equations are similar to that
considered earlier, but the complexity increases considerably (Cook and Cleland, 2007).
A bi-reactant, bi-product reaction may be illustrated by the following:

A + BFP + Q [26]

The reaction might be ordered with A combining with the enzyme (E) before B. The release of products might also be ordered
with P dissociating from the enzyme before Q. Pyruvate kinase (EC 2.7.1.40) catalyzes the following reaction: pyruvate + ATP F
ADP + phosphoenolpyruvate. This enzyme mediates an ordered-sequential bi-bi reaction. Such reactions are diagrammed as
follows (Cleland, 1970):

The reaction might be random (either A or B could combine with the enzyme, and either P or Q could dissociate from the
enzyme). Adenylate kinase (EC 2.7.4.3) catalyzes the following reaction: ATP + AMP F ADP + ADP. This enzyme mediates a
random-ordered bi-bi sequential reaction. This type of reaction is depicted as follows:

One property of both the random-ordered and the compulsory-ordered reaction is that a ternary complex involving the two
substrates and the enzyme form prior to the release of any products.
In contrast to the random- or compulsory-ordered reaction, in ping-pong or double-displacement reactions one product is
released prior to the binding of the second substrate. Thus, the first substrate binds to the enzyme, transfers a piece of itself to the
enzyme, and dissociates as the first product before the second reactant binds. The second reactant picks up the transferred piece and
dissociates as the second product. Aspartate aminotransferase (EC 2.6.1.1) and other transaminases display ping-pong
6 Michaelis-Menten Kinetics

mechanisms. Aspartate reacts with the enzyme, transfers its amino group to the pyridoxal phosphate cofactor of the enzyme, and
dissociates as oxaloacetate. Then a-ketoglutarate binds to the modified enzyme, picks up the amino group, and dissociates as
glutamate. This third major type of bimolecular reaction is depicted in the following diagram where E0 indicates that the enzyme
exists in a chemically modified form.

Steady-State Rate Equations for Enzymes with Two Substrates and Two Products

Many enzymes with two or more substrates obey the Michaelis-Menten equation with respect to one substrate at constant
concentrations of the other substrates. The velocity of the enzyme-catalyzed reaction, where one substrate is varied and the others
are held constant, is represented by a rectangular hyperbola. Enzymes with two substrates (A and B) that exhibit random-ordered or
compulsory-ordered reactions with the formation of a ternary complex obey the following rate equation:

v ¼ Vmax ½A ½B=ðKia Kb + Kb ½A  + Ka ½B + ½A ½BÞ [27]

where Vmax is the maximal velocity with both A and B saturating, Ka is the concentration of A that gives 1/2 Vmax when B is
saturating, Kb is the concentration of B that gives 1/2 Vmax when A is saturating, and Kia is the dissociation constant of the enzyme
for A ([EA] F [E] + [A]).
The corresponding Lineweaver-Burk double reciprocal equation is given by the following formulation:

1=v ¼ Ka =Vmax ð1 + Kia Kb =Ka ½BÞ  1=½A  + 1=Vmax ð1 + Kb =½BÞ [28]

When double reciprocal plots (1/v versus 1/[A] at several fixed concentrations of B) of data for random ordered or compulsory
ordered reactions are examined, a series of lines that intersect to the left of the x-axis result (Figure 3). The lines can intersect above,
on, or below the x-axis. To determine whether the reaction is ordered or random requires additional studies using product
inhibition and other ancillary work.
Secondary plots of the slope and intercept (Figure 4) of the data depicted in Figure 3 provide a means to determine the Vmax
(both [A] and [B] saturating) and other kinetic constants (Ka, Kb, Kia) describing sequential enzyme kinetics.
For enzymes that obey ping-pong (double-displacement) kinetics, the corresponding rate is given by the following equation:

v ¼ Vmax ½A ½B=ðKb ½A  + Ka ½B + ½A ½BÞ [29]

The corresponding Lineweaver-Burk equations are given by the following formulations:

1=v ¼ Ka =Vmax  1=½A  + 1=Vmax  ð1 + Kb =½BÞ [30]

1=v ¼ Kb =Vmax  1=½B + 1=Vmax  ð1 + Ka =½A Þ [31]
When double reciprocal plots (1/v versus 1/[A] at several fixed concentrations of B) of data for ping-pong reactions are
examined, a series of parallel lines is observed. Provided the lines are parallel and do not intersect far to the left of the y-axis, a
diagnosis of a ping-pong mechanism can be made based on the finding of such parallel line plots (Figure 5(a)). Double reciprocal
plots of 1/v versus 1/[B] at several fixed concentrations of B yield similar data (Figure 5(b)).

Figure 3 Lineweaver-Burk plot for an enzyme displaying sequential enzyme kinetics.

 Michaelis-Menten Kinetics 7

Figure 4 Secondary plots corresponding to a sequential enzyme mechanism.

Figure 5 Lineweaver-Burk plot for an enzyme that exhibits ping pong kinetics. In panel (A), A is the varied substrate concentration at several fixed
concentrations of B. In panel (B), B is the varied substrate at several fixed concentrations of A.

Figure 6 Secondary plots for an enzyme exhibits ping pong kinetics. In panel (A), the intercept is plotted as a function of 1/[B]. In panel (B), the
intercept is plotted as a function of 1/[A].

Ka, Kb, and Vmax can be determined by secondary plots of the reciprocal of the substrate concentration versus the intercepts, as
illustrated in Figure 6.

Determination of the Kinetic Constants for Enzyme-catalyzed Reactions

Enzyme kinetic constants (Km and Vmax) are determined using initial velocity measurements at varying substrate concentrations. All
conditions (enzyme concentration, ionic strength, pH, temperature) are kept constant. Only the substrate concentration is varied.
To obtain a wide range of velocities, the substrates are usually varied from 0.25 to 5 Km values, as determined in pilot experiments.
Historically, the enzyme kinetic constants were determined graphically using linear plots and secondary plots as outlined above.
Today, kinetic constants are determined by computer using various programs such as SigmaPlot or GraphPad Prism; the Km and Vmax
values determined in this fashion are used to draw the Lineweaver-Burk lines of ‘best fit’ for illustrative purposes in journal articles.
In the laboratory, plotting the substrate concentration versus velocity, and 1/[A] versus 1/v, provide an indication of trends and may
indicate spurious experimental points. The procedure for determining kinetic constants from Lineweaver-Burk and secondary plots
was described in detail to provide the fundamentals so that the reader can understand the problem as well as the computer can.

Enzyme Inhibitors

The study of enzyme inhibitors can provide information on the mechanism of enzyme catalysis. Moreover, a large number of
drugs inhibit enzymes, and studies of the interaction of drugs with their target enzymes provide clues on the mechanisms leading
to enzyme inhibition. Enzyme inhibitors are divided into two classes: irreversible and reversible. Irreversible inhibitors generally
bind covalently to their target enzymes and inhibit activity for the life of the enzyme.
8 Michaelis-Menten Kinetics

Penicillin, aspirin, and afatinib are examples of irreversible enzyme inhibitors. Penicillin reacts with a bacterial transpeptidase
that participates in cell wall biosynthesis and thereby blocks subsequent catalytic reactions and bacterial growth. Aspirin has been
used therapeutically since the beginning of the 20th century. It forms a covalent adduct with Ser530 of cyclooxygenase I, which
leads to its inhibition (a discovery not made until the 1970s). Cyclooxygenase II also reacts with aspirin, but its active site is larger
and is able to accommodate its substrates and is not inhibited by aspirin. About 10% of non-small cell lung cancers are driven by
mutations in the epidermal growth factor receptor, and this mutant enzyme is inhibited by several FDA-approved protein kinase
inhibitors (www.brimr.org/PKI/PKIs.htm). Afatinib binds to the ATP-binding pocket of EGFR and forms a covalent adduct with a
nearby cysteine residue, which leads to irreversible enzyme inhibition and a therapeutic response (Roskoski, 2014).
Classical reversible enzyme inhibitors bind nearly instantaneously (millisecond time scale) to the active sites of enzymes to exert
their inhibitory effect. These inhibitors do not form a covalent bond with their target enzyme. Reversible inhibitors are divided into
three classes: competitive, uncompetitive, and non-competitive. These inhibitors work by a variety of mechanisms that can be
distinguished by steady-state enzyme kinetics. The initial velocities of an enzyme reaction are measured at several substrate
concentrations without and with fixed concentrations of inhibitor. The data are plotted as the reciprocal of the substrate
concentration versus the reciprocal of the velocities. The key component to examine in such Lineweaver-Burk plots is 1/Vmax.
In competitive inhibition, the inhibitor and a substrate cannot bind to the enzyme simultaneously because they bind to the
same enzyme forms. Competitive inhibitors bind to the same site as the substrate. Increasing the substrate concentration overrides
the inhibitory effect, and at infinite substrate concentration inhibition is nil and 1/Vmax or the y-intercept is unchanged while the
slopes increase with increasing inhibitor concentrations (Figure 7(a)).
In uncompetitive inhibition, the inhibitor can bind to only the enzyme-substrate complex. With increasing inhibitor concen-
trations, the slopes are unchanged, the y-intercepts increase (the Vmax values decrease), and the Michaelis constants decrease (1/
Km values become more negative) as illustrated in Figure 7(b). It is not easy to imagine how this might occur with an enzyme with a
single substrate, but uncompetitive inhibition occurs with multisubstrate enzymes that bind substrates and inhibitors in an
obligatory order. Consider the case of an ordered-sequential reaction with A and B as substrates and P and Q as products as
diagramed above. Under conditions with B saturating, P (an analogue of P) is an uncompetitive inhibitor. P can bind to EQ, an
enzyme-substrate complex, and prevent the release of Q and thus inhibit the reaction. Saturating B prevents the release of B from
EAB and maintains the rightward progress of the reaction.
In non-competitive inhibition, the inhibitor can bind to the free enzyme and to the enzyme-substrate complex. Increasing the
concentration of substrate does not override the binding of inhibitor to the enzyme. The Vmax is decreased while 1/Vmax is
increased. Non-competitive inhibitors have an effect on both the slopes and y-intercepts of the double-reciprocal plots
(Figure 8). The inhibitor may not bind with the same affinity to the enzyme and to the enzyme-substrate complex. In such cases
the lines will intersect above the x-axis (Kis > Kii) or below the x-axis (Kii > Kis) where Kis is the Ki representing the slope effect (the
inhibitor binds to the enzyme) and Kii is the Ki representing the intercept effect (the inhibitor binds to the enzyme substrate
complex). When the inhibitor affinity for the enzyme and enzyme-substrate complex is the same, the lines intersect on the x-axis.
Figure 9 illustrates the interaction of inhibitor with the enzyme. For competitive inhibition with an effect only on the slope of
the Lineweaver-Burk plot, the inhibitor binds to the same form of enzyme as the substrate with which it completes. For

Figure 7 Lineweaver-Burk plots of (A) competitive and (B) uncompetitive enzyme inhibition.

Figure 8 Lineweaver-Burk plots of non-competitive enzyme inhibition.

 Michaelis-Menten Kinetics 9

Figure 9 Enzyme inhibitors and Lineweaver-Burk slope and intercept effects.

uncompetitive inhibition with an effect only on the intercept, the inhibitor binds to the same form of the enzyme as the substrate.
For non-competitive inhibition with an effect on both the slope and intercept, the inhibitor binds to the same form of the enzyme
as the substrate and to the enzyme-substrate complex.
If we have a bi-bi sequential reaction and an inhibitor that competes only with substrate A, when [B] and [I] are kept at a fixed
values and we vary [A], the plot will exhibit only a slope effect indicating that the inhibitor is competitive with respect to A. In this
same situation, when [A] and [I] are kept at fixed values and we vary [B], the double-reciprocal plot with exhibit slope and intercept
effects indicating the inhibitor is non-competitive with respect to B because I does not compete with B. Enzymes with varying
numbers of substrates, products, ordered or random mechanisms, and various inhibitors are described by hundreds of kinetic
mechanisms, which are described in the classic book of Segel (1975). Cleland has described general approaches in deciphering the
numerous mechanisms of enzyme inhibition (Cleland, 1963b).
Several enzymes that serve as therapeutic drug targets are subject to reversible time-dependent inhibition by substrate analogues.
Time-dependent, or slow-binding, enzyme inhibitors, in contrast to classical competitive inhibitors, require seconds-to-minutes to
exert their inhibitory effect. In contrast, classical competitive inhibitors bind instantaneously (millisecond time scale) to the active
sites of enzymes to exert their inhibitory effect. The theoretical advantage of slow-binding inhibitors is not that they take seconds-
to-minutes to inhibit their target enzyme. Rather, after inhibition occurs, it takes minutes-to-hours for the inhibitor to spontane-
ously dissociate from the enzyme, and this reversal cannot be accelerated by substrate. With classical competitive inhibitors, an
increase in substrate concentration due to decreased metabolism can overcome inhibition.
Examples of slow-binding inhibitors and their target enzymes include methotrexate and dihydrofolate reductase, enalopril and
angiotensin-converting enzyme, allopurinol and xanthine: NAD+ oxidoreductase, lovastatin and hydroxymethylglutaryl-CoA
reductase, and indomethacin and prostaglandin H synthases 1 and 2 (Roskoski and Ritchie, 2001). Slow-binding inhibitors of
enzymes dissociate from their target slowly, and this can be therapeutically advantageous. Morrison and Walsh (1988) described
the kinetic characterization of slow-binding inhibitors.

References
Journal Citations

Cleland WW (1963a) Computer programmes for processing enzyme kinetic data. Nature 198: 463–465.
Cleland WW (1963b) The kinetics of enzyme-catalyzed reactions with two of more substrates and products III: prediction of initial velocity and inhibition patterns by inspection.
Biochimica et Biophysica Acta 67: 188–196.
Morrison JF and Walsh CT (1988) The behavior and significance of slow-binding enzyme inhibitors. Advances in Enzymology and Related Areas of Molecular Biology 61: 201–301.
Roskoski R Jr (2014) The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacological Research 79: 34–74.
Roskoski R Jr and Ritchie PA (2001) Time-dependent inhibition of protein farnesyltransferase by a benzodiazepine peptide mimetic. Biochemistry 40: 9329–9335.
Book Citations

Cleland WW (1970) Steady state kinetics. In: Boyer PD (ed.). The enzymes, 3rd ed., vol. 2, pp. 1–65. San Diego: Academic Press.
Cook PF and Cleland WW (2007) Enzyme kinetics and mechanism. New York: Garland Science.
Segel IH (1975) Enzyme kinetics. New York: John Wiley & Sons, This book is a compendium and contains the rate equations corresponding to nearly all theoretical enzyme kinetic
mechanisms, and there is an reprinted edition published by Dover Publications, New York.

Relevant Websites
http://www.brimr.org/PKI/PKIs.htm – This site lists all of the currently FDA-approved protein kinase inhibitors used in the treatment of neoplasms.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html – A site that covers the basics of enzyme kinetics.