ENZYME KINETICS

Medical Biochemistry, Lecture 24

Lecture 24, Outline

Michaelis-Menten kinetics
Interpretations and uses of the MichaelisMenten equation
Enzyme inhibitors: types and kinetics

Enzyme Kinetics Equation

Michaelis-Menten Equation

Initial Velocity (vo) and [S]

The concentration of substrate [S] present will greatly
influence the rate of product formation, termed the velocity
(v) of a reaction. Studying the effects of [S] on the
velocity of a reaction is complicated by the reversibility of
enzyme reactions, e.g. conversion of product back to
substrate. To overcome this problem, the use of initial
velocity (vo) measurements are used. At the start of a
reaction, [S] is in large excess of [P], thus the initial
velocity of the reaction will be dependent on substrate
concentration

Michaelis-Menten Curve

Initial Velocity (vo) and [S]

(cont)
When initial velocity is plotted against [S],
a hyperbolic curve results, where Vmax
represents the maximum reaction velocity.
At this point in the reaction, if [S] >> E, all
available enzyme is "saturated" with bound
substrate, meaning only the ES complex is
present.

Michaelis-Menten Curve

Substrate Saturation of an Enzyme

A. Low [S]

B. 50% [S] or Km C. High, saturating [S]

Steady State Assumption

The M-M equation was derived in part by making several
assumptions. An important one was: the concentration of
substrate must be much greater than the enzyme
concentration. In the situation where [S] >> [E] and at initial
velocity rates, it is assumed that the changes in the concentration
of the intermediate ES complex are very small over time (v o).
This condition is termed a steady-state rate, and is referred to as
steady-state kinetics. Therefore, it follows that the rate of ES
formation will be equal to the rate ES breakdown.

Michaelis-Menten Equation
Derivation

Rate of ES formation = k1([ET] - [ES])

[S] (where [ET] is total concentration of
enzyme E and k-2 is considered neglible)
Rate of ES breakdown to product =
k-1[ES] + k2[ES]

Michaelis-Menten Equation
Derivation (cont)
Thus for the steady state assumption:
k1([ET] - [ES])[S] = k-1[ES] + k2[ES]

This equation is the basis for the final Michaeli

Menten following algebraic rearrangement and
substitution of Km and Vmax terms.

Meaning of Km
An important relationship that can be derived from the
Michaelis-Menten equation is the following: If vo is set
equal to 1/2 Vmax, then the relation Vmax /2 =
Vmax[S]/Km + [S] can be simplied to Km + [S] = 2[S], or Km

= [S]. This means that at one half of the

maximal velocity, the substrate concentration
at this velocity will be equal to the K m. This
relationship has been shown experimentally to be valid for
many enzymes much more complex in regards to the
number of substrates and catalytic steps than the simple
single substrate model used to derive it.

Meaning of Km (cont)
The significance of Km will change based on the different
rate constants and which step is the slowest (also called
the rate-limiting step). In the simplest assumption, the
rate of ES breakdown to product (k 2) is the ratedetermining step of the reaction, so k -1 >> k2 and Km = k1/k1. This relation is also called a dissociation constant for
the ES complex and can be used as a relative measure of
the affinity of a substrate for an enzyme (identical to K d).
However if k2 >> k-1 or k2 and k-1 are similar, then Km
remains more complex and cannot be used as a measure
of substrate affinity.

Uses of Km

Experimentally, Km is a useful parameter for characterizing

the number and/or types of substrates that a particular
enzyme will utilize (an example will be discussed). It is
also useful for comparing similar enzymes from different
tissues or different organisms. Also, it is the K m of the ratelimiting enzyme in many of the biochemical metabolic
pathways that determines the amount of product and
overall regulation of a given pathway. Clinically, K m
comparisons are useful for evaluating the effects
mutations have on protein function for some inherited
genetic diseases.

Meaning of Vmax
The values of Vmax will vary widely for different enzymes and can be
used as an indicator of an enzymes catalytic efficiency. It does not find
much clinical use.
There are some enzymes that have been shown to have the following
reaction sequence:

In this situation, the formation of product is dependent on the

breakdown of an enzyme-product complex, and is thus the rate-limiting
step defined by k3.

Derivation of kcat
A more general term has been defined,
termed kcat, to describe enzymes in which
there are multiple catalytic steps and
possible multiple rate-limiting steps. The
Michaelis-Menten equation can be
substituted with kcat

Definition and Use of kcat

The constant, kcat (units of sec-1), is also
called the turnover number because under
saturating substrate conditions, it represents
the number of substrate molecules converted
to product in a given unit of time on a single
enzyme molecule. In practice, kcat values (not
Vmax) are most often used for comparing the
catalytic efficiencies of related enzyme
classes or among different mutant forms of
an enzyme.

Two Substrate Reactions

Many enzyme reactions involve two or more
substrates. Though the Michaelis-Menten
equation was derived from a single substrate to
product reaction, it still can be used
successfully for more complex reactions (by
using kcat).
Random
Ordered
Ping-pong

Two Substrate Reactions (cont)

In random order reactions, the two substrates
do not bind to the enzyme in any given order; it
does not matter which binds first or second.
In ordered reactions, the substrates bind in a
defined sequence, S1 first and S2 second.
These two reactions share a common feature
termed a ternary complex, formed between E,
ES1, ES2 and ES1S2. In this situation, no product is
formed before both substrates bind to form ES 1S2.

Two Substrate Reactions (cont)

Another possibility is that no
ternary complex is formed and the
first substrate S1 is converted to
product P1 before S2 binds. These
types of reactions are termed
ping-pong or double
displacement reactions.

Km and kcat Example:

HSV-1 Thymidine Kinase
A phosphorylation kinase reaction: T (thymidine) +
ATP is converted to TMP (thymidine monophosphate)
+ ADP
In herpesvirus infected cells, this viral encoded TK
phosphorylates the antiviral drug acyclovir; this is the
pharmacological basis of most herpesvirus treatments
In the last 10 years, this approach has been applied to
cancer gene therapies with HSV-TK and ganciclovir

Thymidine Kinetic Constants for

HSV-1 Thymidine Kinase
(ONLY AN EXAMPLE!!)
HSV-1TK

Km (M) kcat (s-1)

kcat / Km

Gln-125 WT

0.9

0.06

67000

Asn-125

20

0.13

6500

Glu-125

0.003

844

Ganciclovir Kinetic Constants for

HSV-1 Thymidine Kinase
(ONLY AN EXAMPLE!)
HSV-TK

Km (M)

kcat (s-1) kcat / Km

Gln-125 WT

69

0.47

6800

Asn-125

50

0.08

1700

Glu-125

473

0.04

82

Lineweaver-Burk (double
reciprocal plot)
If the reciprocal (1/X) of the Michaelis-Menten equation
is done, after algebraic simplification the following
equation results:

This relation is written in the format of the equation for

a straight line, y = mx + b, where y = 1/v o, m (slope)
= Km/Vmax, x = 1/[S] and the y-intercept, b = 1/V max.
When this relation is plotted,the result is a straight line
graph

Lineweaver-Burk (double
reciprocal plot) (cont)

Uses of double reciprocal plot

The x intercept value is equal to
-1/Km. The biggest advantage to
using the double reciprocal plot is a
more accurate determination of Vmax,
and hence Km. It is also useful in
characterizing the effects of enzyme
inhibitors and distinguishing between
different enzyme mechanisms.

Enzyme Inhibitor Types

Inhibitors of enzymes are generally molecules
which resemble or mimic a particular enzymes
substrate(s). Therefore, it is not surprising
that many therapeutic drugs are some type of
enzyme inhibitor. The modes and types of
inhibitors have been classified by their kinetic
activities and sites of actions. These include
Reversible Competitive Inhibitors, Reversible NonCompetitive Inhibitors, and Irreversible Inhibitors

Definition of Ki
For reversible inhibitors, a term Ki can be
determined.
For competitive inhibitors, the following relation can
be used: Km + I = Km (1 + [I] / Ki ) ;
(where Km + I
is the determined Km in the presence of [I]).
Determining the Ki for other inhibitor types is related
but much more complex and not within the scope of
this lecture or course

Uses of Ki
Ki values are used to characterize and compare
the effectiveness of inhibitors relative to K m. This
parameter is especially useful and important in
evaluating the potential therapeutic value of
inhibitors (drugs) of a given enzyme reaction. For
example, Ki values are used for comparison of the
different types of HIV protease inhibitors. In
general, the lower the Ki value, the tighter
the binding, and hence the more effective an
inhibitor is.

Competitive Inhibition

Vmax - No change
Km INCREASES - indicates a direct interac
of the inhibitor in the active site

Reversible Competitive
Inhibition
Competitive inhibitors compete with the substrate
for binding at the active site (as E + I). In the
double reciprocal plot for a competitive inhibitor
acting at the substrate site for the following
reasons, notice with increasing concentration of
inhibitor, the Vmax does not change; however,
the Km of the substrate is increased. This also
reflects the reversible nature of the inhibitor; there
is always some concentration of substrate which
can displace the inhibitor.

Non-Competitive Inhibition

Vmax DECREASES - inhibitor affects rate of re

by binding to site other than substrate active
Km - No change

Reversible Non-Competitive
Inhibition
Non-competitive inhibitors combine with both the enzyme (E
+ I) and the enzyme-substrate (EI + S) complex. The inhibitor
binds to a site other that the substrate site, and is thus
independent of the presence or absence of substrate. This
action results in a conformational change in the protein that
affects a catalytic step and hence decreases or eliminates
enzyme activity (formation of P). Notice in the reciprocal plot,
a non-competitive inhibitor does not affect the binding of the
substrate (Km), but it does result in a decrease in Vmax.
This can be explained by the fact that since inhibitor bound to
an enzyme inactivates it, the more EI formed will lower [ES]
and thus lower the overall rate of the reaction V max.

Irreversible Inhibitors

Irreversible inhibitors generally result in the destruction or

modification of an essential amino acid required for enzyme
activity. Frequently, this is due to some type of covalent link
between enzyme and inhibitor. These types of inhibitors range
from fairly simple, broadly reacting chemical modifying reagents
(like iodoacetamide that reacts with cysteines) to complex
inhibitors that interact specifically and irreversibly with active site
amino acids. (termed suicide inhibitors). These inhibitors are
designed to mimic the natural substrate in recognition and binding
to an enzyme active site. Upon binding and some catalytic
modification, a highly reactive inhibitor product is formed that
binds irreversibly and inactivates the enzyme. Use of suicide
inhibitors have proven to be very clinically effective

Irreversible Inhibitor: Allopurinol

Irreversible Inhibitor: Penicillin (Ex)

Diisopropyl Phosphofluoridate:
Irreversible Acetylcholinesterase
Inhibitor (Example)

Inhibitor Summary
REMEMBER - The types of enzyme inhibitors
described have been for relatively simple,
single substrate-product reactions that obey
Michaelis-Menten kinetics. However, not all
enzyme inhibitors will necessarily be one type
of inhibitor. Especially for some multisubstrate reactions, a particular inhibitor can
be competitive for one substrate and noncompetitive with a second or third substrate.
Also, suicide inhibitors by design are generally
competitive inhibitors of a substrate, and
therefore must first bind in the active site.