ENZYME KINETICS

Medical Biochemistry, Lecture 24

 Lecture 24, Outline
• Michaelis-Menten kinetics

• Interpretations and uses of the Michaelis-

Menten 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 Michaelis-

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 Km.
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 (k2) is the rate-determining step of the
reaction, so k-1 >> k2 and Km = k-1/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 Kd). 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 Km of the rate-limiting enzyme in many
of the biochemical metabolic pathways that determines the amount
of product and overall regulation of a given pathway. Clinically,
Km 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 k 3.
 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 ES1S2.
 Two Substrate Reactions (cont)
• Another possibility is that no ternary
complex is formed and the first substrate S 1
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!!)
-1
HSV-1TK Km (M) kcat (s ) kcat / Km

Gln-125 WT 0.9 0.06 67000

Asn-125 20 0.13 6500

Glu-125 3 0.003 844

Ganciclovir Kinetic Constants for
HSV-1 Thymidine Kinase
(ONLY AN EXAMPLE!)
-1
HSV-TK Km (M) kcat (s ) 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/vo, m (slope) = Km/Vmax,
x = 1/[S] and the y-intercept, b = 1/Vmax. 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 Non-Competitive 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 Km. 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 interaction
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 reaction

by binding to site other than substrate active-site
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 Vmax.
 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 multi-substrate reactions, a particular
inhibitor can be competitive for one substrate and
non-competitive 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.