ENZYMES: Kinetics
- Field of Biochemistry concerned
with
o The quantitative
measurement of the rates of
enzyme-catalyzed reactions
o The systematic study of
factors that affect these rate
Homeostatis - balanced, coordinated
Enzyme kinetics
- Permit the reconstruction of the
number and order of the individual
steps which enzymes transform
substrate  products
- Identify potential drugs that
selectively enhance or inhibit the
rates of specific enzyme-catalyzed
processes
- Reveal details of the catalytic
mechanism
- Balanced chemical equation
o Lists the initial chemical
species (substrates) present
and the new chemical species
(products) formed for a
particular reaction
- Stoichiometry
o All in their correct
proportions
- By kinetics and chemical
modification, the central question
about enzymes:
o How do they work?
o How do they catalyze
reactions?
- A typical overall enzyme-catalyzed
reaction involving a single substrate
and a single product
o E+A↔E+Z
o E = enzyme
o A = substrate
o Z = product
o Double arrows = reaction
occurs in both directions
- Mechanism of enzyme action
o 2 perspectives
 Enzymes provide an
alternate,
energetically
favorable reaction
pathway different
from the uncatalyzed
reaction
 Active site chemically
facilitates catalysis
- Gibbs Free Energy (∆G)
o Direction in which a
chemical reaction will tend to
proceed
o Concentrations of reactants
and products that will be
present in equilibrium
o ∆ Gp - ∆Gs = ∆G
o ∆G0 : free energy that
accompanies transition from
the standard state, one-molar
concentrations of S & P, to
equilibrium
- ∆G0’ : ∆G0 at a standard state of 10
-7 M protons at pH 7
- Negative : if the free energy of the
products is lower than that of the
substrates
- Direction left to right
- SPONTANEOUS
- Sign and magnitude of free energy
change determine how far the
reaction will proceed
- ∆G0
o Independent of the
mechanism of reaction
o Provide information only
about the direction and
equilibrium state of the
reaction
o Provides no information
concerning the rates of
reaction
- Virtually all chemical reactions have
an energy barrier separating the
reactants and the products
- Barrier: free energy of activation
- Energy difference between that of
the reactants and a high-energy
intermediate that occurs during the
formation of the product
- Rate of reaction
o Sufficient energy needed for
molecules to react
o Energy to overcome the
energy barrier of the
transition state
o No enzymes  only a small
portion of the molecules may
possess enough energy to
achieve the transition state
between reactants and
products
o Determined by the number of
energized molecules
o The lower the free energy of
activation  the more
molecules have sufficient
energy to pass through the
transition state  the faster
the rate of reaction
- Transition state
o A reaction may be looked at
as passing from one valley,
representing stable reactants,
over a mountain pass to
another valley, the products.
o The pass between them is
called the transition state,
the state from which the
molecules may with equal
probability go ahead to
products or back to reactants.
o Energy must be put into a
reaction to raise the reactants
to the top of the pass, the
transition state; this energy is
called the free energy of
activation or activation
energy
o The role of the catalyst is to
find a lower pass over the
mountain range, a pathway
with a lower activation
energy
o An alternative way of looking
at the role of a catalyst is that
it stabilizes the transition
state
- Alternate reaction pathway
o Provides the enzyme
allowing a reaction to
proceed rapidly under
conditions prevailing in the
cell
o Condition: lower free energy
of activation
o Enzyme does not change the
free energies of the reactants
or products and does not
change the equilibrium of the
reaction
Kinetic theory
- Collision theory
o For 2 molecules to react they
must
 Approach within
bond-forming
distance of one
another (collide)
 Possess sufficient
kinetic energy to
overcome the energy
barrier for reaching
the transition state
Factors that affect the reaction rate
- Temperature
o ↑ temperature = ↑ kinetic
energy of molecules
- Reactant concentration
o ↑ concentration = ↑ frequency
with which molecules collide
o A+BP
o Concentrations of either A &
B are doubled  probability
of collision will double
o If both are doubled 
increase is fourfold
Keq : ratio of rate constants
- At equilibrium, the overall
concentrations of reactants and
products remain constant
- The rate of conversion of substrates
to products therefore equals the rate
at which products are converted to
substrates
Equilibrium
- Equilibrium constant is a ratio of the
reaction rate constants
- Reaction rates of the forward and
back reactions are equal
- Dynamic state
- No net change in the concentration
of S & P
- Individual S & P molecules are
continually being interconverted
- Numeric value of the equilibrium
constant Keq can be calculated
o From the concentration of S
& P at equilibrium
o From the ratio K1/K-1
Enzyme kinetics
- An alternative way of looking at the
role of a catalyst is that it stabilizes
the transition state
- The environment of the active site
lowers ∆Gf by stabilizing the
transition state intermediates
- How?
o Acid-base groups suitably
positioned to transfer protons
to and from the developing
transition state intermediate
o Suitably positioned charged
groups or metal ions that
stabilize developing charges
o Imposition of steric strain on
S so that their geometry
approaches that of the
transition state
Factors that affect the rates of enzyme-
catalyzed reactions
- Temperature
o ↑ temperature  ↑ rate of
both catalyzed and non-
catalyzed reactions by ↑
kinetic energy and collision
frequency of the reacting
molecules
o Heat can also ↑ kinetic
energy of the enzyme 
exceeds the energy barrier for
disrupting their noncovalent
interactions that maintain the
enzyme’s 3-D structure 
DENATURATION
o Denaturation  loss of
catalytic activity
o In humans  stable at 45-
55°C
o Clinical importance: fever or
hypothermia
- Hydrogen ion concentration
o Intracellular enzymes exhibit
optimal activity at pH : 5-9
o Reflects balance between
denaturation at high or low
pH
o Acid-base catalysis : residues
involved must be in the
appropriate state of
protonation for the reaction to
proceed
o Most common charged
groups
 Negative carboxylate
groups
 Protonated amines
o Gain or loss of critical
charged groups adversely
affect substrate binding 
retard or abolish catalysis
pH
- Effect on the ionization of the active
site
o Catalytic activity may require
that an amino group of the
enzyme may be in the
protonated form
 o At alkaline pH  group is
deprotonated  ↓ rate of
reaction
- Effect on enzyme denaturation
o Extremes of pH 
denaturation
o Structure of catalytically
active protein molecule
depends on the ionic
character of the amino acid
side chains
Michaelis-Menten kinetic theory
- Effect of ENZYME
CONCENTRATION on reaction
velocity
o If the substrate concentration
is held constant  velocity of
the reaction in proportional to
the enzyme concentration
- Effect of SUBSTRATE
CONCENTRATION on reaction
velocity
o [S] is low, v is 1st order with
respect to substrate
o [S] ~ v
o [S] is high, reaction is zero-
order (independent of [S])
o Mid-[S], reaction is mixed-
order (proportionality is
changing)
- Plotting Vi as a function [S], we find
that
o At low values of [S], the
initial velocity, Vi, rises
almost linearly with
increasing [S]
o But as [S] increases, the gains
in Vi level off (forming a
rectangular hyperbola)
o The substance concentration
that produces a Vi that is one-
half of Vmax is designated the
Michaelis-Menten constant
o Km is (roughly) an
inverse measure of the affinity or
strength of binding between the
enzyme and its substrate.
o The lower the Km, the
greater the affinity (so the lower the
concentration of the substrate needed
to achieve a given rate).
MICHAELIS-MENTEN KINETIC
THEORY
 Lineweaver-Burk linear plot:
o Used because it is
difficult to estimate
Vmax from the
position of the
asymptote (plot of a
rectangular
hyperbola)
 Plotting the reciprocals of the
same data points yields a
“double-reciprocal” or
Lineweaver-Burk plot. This
provides a more precise way
to determine Vmax and Km.
 Vmax is determined by the
point where the line crosses
the 1/Vi = 0 axis (so the [S] is
infinite).
 Note that the magnitude
represented by the data points
 in this plot decrease from IMPORTANT CONCLUSIONS
lower left to upper right.
 Km equals Vmax times the  Characteristics of the Km
slope of line. This is easily ->
determined from the intercept o Characteristics of
on the X axis. an enzyme and its
substrate
o Reflects the
affinity of the
enzyme for that
substrate
o Numerically equal
to the substrate
concentration at
which the reaction
velocity = ½
Vmax
o Does not vary
with the
concentration of
the enzyme
 3 KEY ASSUMPTIONS
o [S] is very large  Small Km
compared with [E] -> o High affinity of
when all E are bound, the enzyme for the
there is still an excess substrate
of S o Lower
o Only the initial concentration of
velocity conditions the substrate is
are considered -> very needed to half-
little accumulation of saturate the
P -> formation of ES enzyme
from E + P is
negligible  Large Km
o STEADY STATE o Low affinity of
ASSUMPTION: rate the enzyme for the
of breakdown of ES = substrate because
rate of formation of a high
ES concentration of
substrate is
needed to half-
saturate the
enzyme.
  Velocity-Enzyme
Concentration
o Rate of reaction is
directly
proportional to the
enzyme
concentration
o If the enzyme
concentration is
halved -> initial
rate of reaction
and Vmax is
reduced to half
that of the original
ENZYME INHIBITION
 Reversible
o Competitive inhibition
 Inhibitors compete
directly with substrate for
binding to the active site
(i.e., catalytic site)
 Overcome by raising the
concentration of the
substrate
o Uncompetitive inhibition
 Inhibitors bind only to the
ES complex at a site
distinct from the active
site (i.e., the allosteric
site)
o Noncompetitive inhibition
 Inhibitors bind both to the
free enzyme and to the ES
Complex at the allosteric
site, which is distinct
from the active site
 “Pure” noncompetitive
inhibitor
 “Mixed” noncompetitive
inhibitor
 Irreversible Competitive Inhibitors
o Bind covalently so tightly to the
active site that the enzyme is
inactivated irreversibly
o Does not obey the Michaelis-
Menten kinetics
o AFFINITY LABELS
 Substrate analogs that
possess a highly reactive
group that is not present
on the natural substrate
 The active site is
permanently blocked
from the substrate
because the group reacts
covalently with amino
acid residue
 The residue modified –
not necessarily involved
in catalysis
o MECHANISM BASED or
SUICIDE INHIBITORS
 Substrate analogs that are
transformed by the
catalytic action of the
enzyme

 Product of this reaction is
highly reactive ->
combines covalently with
amino acid residue in the
active site -> enzyme
inactivation
o TRANSITION STATE
ANALOGS
 Substrate analogs whose
structures closely
resemble the transition
state of the natural
substrate
 Transition-state analogs
do not covalently modify
the enzyme but bind the
active site so tightly that
 they irreversibly ADDITIONAL NOTES FROM
inactivate it LIPPINCOT

ENZYME INHIBITION

 Provide pharmacologic agents

and research tolls for study of
mechanism of enzyme action
 MEDICAL RELEVANCE
o Highly toxic, naturally-
occurring & man-made
compounds (irreversible
enzyme inhibitors)
o Therapeutic – natural and
synthetic