MODELS FOR ENZYME KINETICS
Why do we study kinetics?
 The study of enzyme kinetics is a powerful way
of investigate an enzyme’s mechanism.
 Leading to a better understanding of general cell
metabolism as well as an understanding of drug
action and the design of medicinal agents.
 Kinetic essay with enzymes are widely used in
drug discovery and development.
Leonor Michaelis- American biochemist of German
origin who studied the catalytic activity of enzymes.
Maud Leonora Menten - a Canadian bio-medical and
medical researcher who made significant contributions to
enzyme kinetics and histochemistry.
MOLECULARITY AND REACTION ORDER OF ENZYME
KINETICS
• Enzyme kinetics has a unusual feature in that, as
the concentration of substrate increases, we
seem to have second-order kinetics that
gradually switches to first-order kinetics
Why does this happen?
 Depending on whether we have mostly free
enzyme or mostly saturated enzyme present in
solution
Low [S] compared to Km = mostly free enzyme
Approximately rate law for Low [S] compared to
Km: Second Order
𝑘+2 [𝐸0 ][𝑆]
𝑅𝑎𝑡𝑒 = 𝑣 =
𝐾𝑚
• High [S] compared to Km = enzyme active sites
are filled with substrate
Approximate relation for High [S] compared to
Km: First Order
𝑅𝑎𝑡𝑒 = 𝑘+2 [𝐸0 ] = 𝑉𝑚𝑎𝑥
UNITS OF ENZYME ACTIVITY
• Traditional unit (symbol U) of enzyme activity is
the amount of enzyme that can convert one
micromole of substrate into products in one
minute
• Katal (symbol kat) : SI unit of activity ; the
amount of activity that converts one ole of
substrate into products in one second
• 1 microkat = 60 (traditional) unit of activity
NUMERICAL VALUES OF KINETIC PARAMETERS
Carbonic anhydrase – CO2
 One of the fastest acting enzyme known.
 Its turnover number is extremely high.
 This enzyme operates essentially under
“diffusion control
 8000 micromolar
Lactate dehydrogenase-pyruvate
 somewhat slower acting but has higher affinity
for its substrate
 140 micromolar
Elastase-Ac-Pro-Ala-Pro-Ala-NH2
 very slow kinetically
 3900 micromolar
Fumrase-Fumarate
 High turnover number and fairly good affinity for
its substrate
 27 micromolar
Interpretation of the michaelis parameter
• This value of enzyme range widely and often
dependent on environmental conditions such as
pH, temperature, and ionic strength.
• The Michaelis Constant, KM is very important in
determining enzyme-substrate interaction.
• The KM is able to detect two factors:
 • One is the concentration of substrate  The initial rate of a reaction is the
when the reaction velocity is half that of instantaneous rate at the start of
the maximal velocity; the reaction (i.e., when t = 0). The initial rate is
equal to the negative of the slope of the curve of
• Secondly, it is, in some cases, able to
reactant concentration versus time at t = 0.
detect the strength of the enzyme-
substrate complex (ES). When, and only Direct Plot
when k2 << k-1, High KM indicates weak
binding and low KM indicates strong  The kinetics of enzymatic reactions can be
binding. described by the Michaelis–Menten equation,
that is, V = Vmax × [S]/([S] + Km), where V is the
[𝐸][𝑆] 𝑘−1 𝑘+2
𝑘𝑆 = = (dissociation) 𝑘𝑀 = 𝑘𝑆 + reaction rate, [S] is the substrate concentration,
[𝐸∙𝑆] 𝑘−2 𝑘+1
and Km is the half-saturation constant. Km and
Vmax are determined by the characteristics of
the enzyme.
Meaning of 𝒌𝒄𝒂𝒕 /𝑲𝒎
• For saturating concentration of substrate, the How to identify Km?
maximal rate reaction is given
𝑉𝑚𝑎𝑥 = 𝑘𝑐𝑎𝑡 [𝐸0 ] 𝑉m[S] 𝑉m[S] 𝑉m[S] 𝑉m
𝑣= 𝑣= 𝑣= 𝑣=
• To saturate the active site, we must have [S] >> Km + [𝑆] [𝑆] + [𝑆] 2 [𝑆] 2
𝑲𝒎
Key points
• Most of the active sites are vacant when the
substrate concentration is low, such as [S] << 𝑲𝒎  Km is a characteristic of each substrate/enzyme
of how attracted they are with each other
• If we neglect the [S] by comparison of 𝑲𝒎 in the
 Can determine Vm/Km from Michaelis-menten
denominator of equation 4 , then the rate
plot and Lineweaver Burk plot
equation is :
 Vm depends on the amount of enzymes present
𝑘𝑐𝑎𝑡
𝑣= [𝐸 ][𝑆]
𝑘𝑀 𝑜
Double-Reciprocal (Lineweaver-Burk) Plot
The ratio Kcat/KM – often referred to as the ‘specificity The double-reciprocal equation is obtained by taking
constant’ – is a useful index for comparing the relative the reciprocal of both sides of the Michaelis-
rates of an enzyme acting on alternative, competing Menten equation. The double-reciprocal (also known as
substrates. However, an alternative description, the Lineweaver-Burk) plot is created by plotting the
‘catalytic efficiency’, is frequently used, and on occasions inverse initial velocity (1/V0) as a function of the inverse
misused, to compare the reactivity of two enzymes of the substrate concentration (1/[S]).
acting on the same substrate.
𝑉m[S] 1 Km [𝑆]
𝑣= = +
Km + [𝑆] 𝑣 𝑉m[S] 𝑉m[S]
Graphical Representations of the Michaelis-Menten
Model 1 Km + [𝑆] 1 1 1
Meaning of: = =𝑐 +
𝑣 𝑉m[S] 𝑣 [S] Vm
[E] = Enzymes (Protein)
[S]= Substrate concentration
V= Rate of reaction
V0 = Initial rate of reaction Reversible Competitive Inhibition
Vmax = Maximum rate of reaction Most inhibitors produce their effects in reducing the
KM = Michaelis constant overall rate of reaction by forming a complex with
enzyme.
Initial Rate of Reaction
 V0=initial rate of reaction
Competitive Inhibitors - compete with substrate for
binding to the enzyme; formation of the [E][S] complex
is reduced while a new type of complex, [E][I] is formed.
 Formation of the [E][I] complex reduces the free
enzyme concentration, and hence the tendency
of the [E][S] complex to form. The net effect is
reduced the rate of reaction.
 In competitive inhibition, a higher concentration
of substrate can restore the original rate of
reaction. The higher [S] corresponds to an
effective increase in kM.
 The concentration of S increases, the equilibrium
of E with [E][S] and [E][I] shifts away from [E][I]
and toward [E][S], which then permits the
catalytic reaction step to occur and so form
product. The maximum velocity Vmax does not
change.
 For competitive inhibition, the double-reciprocal
plot is still linear. The intercept on the vertical
axis is the same, so Vmax has not changed.
However, the slope of the plot increases as I
increases, reflecting the change in the apparent
Michaelis Constant.
Uncompetitive Inhibition
 Principle: the inhibitor binds to the [E][S]
complex, not to the free enzyme, and forms an
[E][S][I] complex. No [E][i]complex is formed.
The [E][S]complex is catalytically inert.
 For uncompetitive inhibition kM is decreased in
magnitude and Vmax is reduced as well, it's not
possible to overcome the inhibition and so reach
Vmax by adding more and more substrate
because it gives the inhibition more
[E][S]complexes to bind to, and more
opportunities to inhibit enzyme; Compare to
competitive inhibition, only kM is affected.
MIXED AND NONCOMPETITIVE INHIBITOR
NONCOMPETITIVE INHIBITOR - is a special case of mixed
inhibition. The inhibitor has the same affinity for either E
or [E][S]complex, that is K1 is numerically equal to K'1.
MIXED INHIBITION - the quantities K1 and K'1 are
allowed to be different.
 The inhibitor I can form a complex both with free
enzyme E and with the enzyme-substrate
complex [E][S]. The [E][I] and [E][S][I] complexes
are both catalytically inactive.
 Because I will bind to the [E][S] complex, it's not
possible to overcome inhibition by adding more
substrate. The Vmax achievable now is
something less than that achieved when no
inhibitor is present.
IRREVERSIBLE INHIBITION
 Generally the irreversible inhibition of an
enzyme entails covalent attachment of the
inhibitor to the enzyme or some covalent
modification, involving the key residues of the
enzyme, by the inhibitor.
 The covalent attachment prevents the inhibitor
from dissociating from the enzyme, hence the
inhibition is considered irreversible.
 EXAMPLE: Aspirin that targets an enzyme
involved in inflammation. It contains a reactive
acetyl group, and the active site of the target
enzyme (prostaglandin H synthase)
SUICIDE SUBSTRATES
 An inhibitor is unreactive until the enzyme
attempts to use it as a substrate. At this point,
the compound binds covalently to the active site
and inhibits the enzyme permanently.
 The enzyme “kills itself” by this sort of reaction;
thus the inhibitor is described as a “suicide
inhibitor”.
 EXAMPLE: Penicilin Is an reactive until the
bacterial transpeptidase accepts it as a
substrate; then becomes covalently linked,
irreversibly, in the enzymes active site.
HEAVY METAL TOXICITY
 Most enzymes have sulfyhydryl groups that are
needed either for catalytic activity or for
structural reasons.
  These groups can form tight bonds with heavy
metals such as mercury, lead, silver, and even
iron and copper.
 Once the heavy metal atom is bound, the
sulfyhydryl cannot function in catalysis. The
presence of metal atom can distort the structure
of the enzyme.
NERVE AGENTS
 Many chemical warefare agents react with
enzymes to inhibit them. Certain organic
phosphates are extremely toxic targeting
acetylcholinesterase.
INTRODUCTION TO DESIGN OF ENZYME INHIBITORS
Enzymes as Drug Targets
 By picking a key enzyme and inhibiting it, one
can reduce the concentration of the
metabolites and/or build up the
concentration of substrates.
 Some examples of such inhibitors currently
in use include the following:
1. Statins inhibit HMG CoA reductase,
leading to lower cholesterol levels
2. Gleevec inhibits a protein kinase and
blocks proliferation of cancer cells.
3. Aspirin inhibits a cyclo-oxygenase
and reduces inflammation.
COMBINATION CHEMOTHERAPY
 In combination chemotherapy, the main idea is
to combine two inhibitors that target the same
pathway to achieve an enchanced response.
 An example is a one very effective antibacterial
combination, trimethoprim and
sulfamethoxazole forming a combination called
co-trimoxazole.
 A variation on this idea is to use one compound
to protect the other from metabolic
degradation.
EXAMPLES OF INHIBITOR DESIGN
Four basic strategies can be used in inhibitor design:
mimic the substrate, mimic the transition state;
irreversibly react with a key residue; or change the
enzyme conformation or state of assembly
1. Substrate mimics
 Substrate mimics are usually
competitive inhibitors.
 The strategy here is to use compounds
that have much but not all of the
chemical feature of the substrate, so
that they fit into the active site and are
held relatively tightly
2. Transition state mimics
 Compounds can be made that resemble
the transition state but that cannot go
on to complete the reaction.
 These compounds should bind more
tightly to the enzyme then does the
natural substrate, given that one way in
which enzymes function is to stabilize
the transition state by binding tightly to
it.
3. Irreversible Inhibition
 A compound with good affinity for the
enzyme may be used that contains a
reactive group that can link covalently
and irreversibly to the enzyme.
4. Uncompetitive Inhibitors
 A classical uncompetitive inhibitor forms
a ternary complex with the enzyme and
the substrate, and blocks turnover of the
substrate into the product. At the same
time, the inhibitor forms negligible
amounts of complex with the free
enzyme