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Enzyme Kinetics
S.M.K
10
10//2010
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 Enzymes background
 Definition

 Important prosperities

 Enzyme Kinetics
 Michaelis--Menten equation
Michaelis
 Line weaver-
weaver-Burk plot
 Practical exercise
 Enzymes background
 Enzyme definition
Enzymes are the catalysts of biological systems
and are extremely efficient and specific as catalysts.

 Prosperities of Enzymes
 Accelerates the rate of the reaction

 Lower the activation energy of the reaction

 Highly specific
 Enzyme accelerates the of a reaction
 Enzyme accelerates the rate of a reaction by factors of at least a million compared to
the rate of the same reaction in the absence of the enzyme without affecting the
equilibrium.
 Enzymes do not alter the equilibrium point of the reaction. This means that the
enzyme accelerates the forward and reverse reaction by precisely the same factor.
åor exampleü consider the interconversion of A and B.
AȼB
Suppose that in the absence of the enzyme the forward rate constant (kf) is 10-4
s-1 and the reverse rate constant (kr) is 10-6 s-1. The equilibrium constant (Keq) is
given by the ratio of the two rate constants.

 The equilibrium concentration of B is 100 times that of A whether or not an enzyme is

present. Howeverü in the absence of an enzyme the reaction could take more than an
hour to approach this equilibriumü whereas in the presence of an enzymeü equilibrium
could be attained within a 2 second.
 Enzyme lowers the activation energy of reaction
 The enzyme lowers the height of the energy barrier to the reaction thereby
increasing the rate of the reactionü but since the rate of both the forward and
reverse reaction are affected by the same amountü the equilibrium constant is
not affected by the presence of the enzyme.
 EAf is the activation energy for the forward reaction in the absence of a catalyst
and E·Af is the activation
energy forthe forward reaction
in the presence of a catalystü

ƦGo is the change in free

energy for the reaction.Since
ƦGo is the same for the catalyzed
and uncatalyzed reactionü Keq is the same for both reactions.
 Enzymes are highly specific

 Enzymes are highly specific. Typically a particular enzyme catalyzes only a

single chemical reaction or a set of closely related chemical reactions.

 There are two proposed models to explain the specificity of the interaction
between the substrate molecule and the active site of an enzyme.
(a) The ´lock and keyµ model ² in this model the substrate has a shape
matching the enzyme·s active site
 (b) The ´induced fit modelµ ² the active site has a shape
complementary to that of the substrate ½


½
 

 Enzyme kinetics
 Gefinition
It is the branch of enzymology that deals with the factors
affecting the rates of enzyme-
enzyme-catalyzed reactions.

 åactors affecting enzyme reaction

Enzyme concentration
Ligand concentration (substrate, products, inhibitors and activators)
Temperature
pH
Ionic strength
 Effect of enzyme concentration
 §ate of the enzyme catalyzed reaction depends directly on the
concentration of the enzyme [E]
in the presence of an excess of the compound which is being
transformed (substrate)

Velocity (V)= d[p]/d[t]

 Effect of substrate concentration
 At fixed concentration of enzyme
an increase of substrate will result inïï
At first : a very rapid rise of velocity or reaction rate
Then the increase in the rate of the reaction begins to slow down until
at a large substrate concentration no further increase in the velocity

By plotting the velocity (rate of reaction ) against substrate

concentration a section of rectangular hyperbola is obtained

Vedio (1) (2
(2)
This curve is biphasic
 Phase 1: (1st order kinetics)

 Phase 2: (zero order kinetics)

Phase 1: there is low substrate concentrations ï the active site

on the enzyme molecules are not saturated by the substrate
= §eaction rate is depending on substrate concentration
ï ( 1st order kinetics)
Phase 2 : the number of substrate molecules increasesü ï
the sites are covered to a greater extended until reach
saturation no more sites are available ï The Enzyme is
working at full capacity
=§eaction rate is independent of substrate concentration
ï( zero order kinetics)
Michaelis--menten equation
Michaelis
 The mathematical equation the define the
quantitative relationship between the rate of the
reaction and substrate concentration

V is the observed velocity at given substrate

concentration [s]; Km represent the amount of
substrate required to bind with half of the
available enzyme and produce half of maximal
velocity V max -

 Votes

ï if [s] is very large ([s]>100 km) ü Km become

insignificant and V= V max
ï If V=1/2 Vmax ü Km=[s]
ï If [s] is very small ([s]<0.01 Km) ü equation will be
V =( Vmax [s])/Km
 Mraphical determination of Vmax and Km
 Because the graphical drawing of V versus [s] is
hyperbola it is difficult to determine Km and Vmax
 So we use line weaver-
weaver-burk plotü where 1/[v] versus
1/[s] derived from michaelis-
michaelis- menten equation
 Votes
ï The substrate concentration chosen to generate
the plot should be in the neighborhood of Km
([s] in the range from 0.33 to 2 Km)

ïIf [s]=3.3 to 20Km the obtained curve will be

horizontal
ïAlso if [s] is very small ([s]=0.033-
([s]=0.033-0.2 Km)
curve will intercept both axis too close to the
origin Km or Vmax cannot be determine
accurately