Enzymes Continued

Lecture 3
Fall 2007
 Experimental Determination of
Rate Parameters
• Non-linear equation – goal is to linearize it
• Double Reciprocal or Line-Weaver Burke Plot
1 1 K 1 Plot 1/v vs 1/S
= + M
v Vmax Vmax S Separates v and S
Low S bias
• Eadie- Hofstee Plot
v Plot v vs v/S
v = Vmax − K M
S Less bias on low S
• Hanes Wolf Plot
S KM 1 Plot S/v vs S
= + S
v Vmax Vmax More accurate Vmax
 Example Plots
Line
Weaver
Burke

Hanes
Wolf

Eadie
Hofstee
 More Complex Enzyme Models
• MM does not describe every substrate enzyme rxn, although it does a great
job for many.
• Allosteric enzymes - more than one substrate binding site, binding of one
substrate facilitates binding of another substrate molecule - cooperative
binding.

dS vmax S n
v=− =
• n = cooperativity coeff. and dt K M + positive
n > 1 indicates S n
cooperativity.
 More Complex Enzyme Models
• Reversible Product Formation
S + E <----------> ES <-----------> E + P
• More than one Substrate
• Enzyme Inhibition
– metal ions
– high concentrations of substrate or product
– other organic molecules
 Enzyme Inhibition Models
Competitive
Competitive Inhibition - substrate and inhibitor compete for the enzyme
S + E <------------> ES -----------> E + P (1)
k1 k3
E + I <--------> EI (2)
k2
then = K kand M
4 = K dissociation constants
I

- check ratio for

k5 Ki
k2 k4
k1 k3
"KM" is effected in competitive inhibition v=maxKMS(1 + i/KI)
v= KM, app

 i 
S + K M 1 + 
 K I 
 Enzyme Inhibition Models
Non-competitive
Noncompetitive Inhibition - inhibitor and substrate bind
simultaneously to enzyme, binding of one does not influence
the affinity of either species to complex with the enzyme.

S + E <------------> ES -----------> E + P
E + I <-------->
KM EI dissociation constant KI
EI + S <-------->
K EIS dissociation constant KM
I
ES + I <-------->
KM EIS dissociation constant KI

KI
 Enzyme Inhibition Models
Non-competitive
[ E ][ S ] [ EI ][ S ]
KM = =
[ ES ] [ ESI ]
[ E ][ I ] [ ES ][ I ]
KI = =
[ EI ] [ ESI ] The maximum
 vmax S  velocity is affected
1 + ( I / K )  vmax S
v=  I 
vmax,app =
S + KM 1+ ( I / KI )
 Enzyme Inhibition Models
Uncompetitive
Uncompetitive Inhibition - inhibitors bind to the
enzyme substrate complex but not the enzyme itself.
KM
S + E <------------> ES -----------> E + P
KI
ES + I <--------> ESI dissociation constant KI
Vm
S
1+ I / KI Vmax,app S
v= =
KM K + S
+S m , app
1+ I / KI
 Enzyme Inhibition Models
Substrate Inhibition
Substrate Inhibition - too much substrate
S + E <------------> ES -----------> E + P
ES + S <---------> ES2 dissociation constant KS1

vmax S vmax S
v= =
S2 KM S
KM + S + KM + +
K S1 S K S1
 Enzyme Inhibition Models
Substrate Inhibition
• Low substrate concentration S2/KS1 << 1 no
inhibition observed
vmax S
v=
KM + S
• High substrate concentration KM/S << 1
where inhibition dominates
vmax
v=
 S 
1 + 
 K S1 
 Enzyme Inhibition
• Can have any combination of effects.
• The "Mixed" case on Handout is a
combination of noncompetitive and
competitive inhibition.
 Class Exercise
Problem 3.5 in text
An Inhibitor is added to the enzymatic reaction at a level
of 1.0 g/L. The following data were obtained for KM= 9.2 g
S/L
v S
0.91 20
0.66 10
0.49 6.67 A) Is the inhibitor competitive
0.4 5
0.33 4 or noncompetitive?
0.29 3.33
0.23 2.5
B) Find KI.
Other Things that Affect Enzymes
– Focus on Binding Site
Other Things that affect enzymes

• pH
• Temperature
• Fluid forces - hydrodynamic forces, hydrostatic
pressure and interfacial tension
• Chemical agents (alcohol, urea and hydrogen
peroxide)
• Irradiation (light, sound, ionizing radiation)
 pH

pH = log10 (1/[H+]) = - log10 [H+]

Ionization equilibrium of an acid

HA <-----> H+ + A-
Equilibrium constant + −
[ H ][ A ]
K=
The pK of an acid is defined as [ HA]
pK = - log K = log (1/K)
 pH Effects
• Variations in pH effect the ionic form of the active site, changing the enzyme activity
and thus the reaction rate.
Since enzymes contain amino acids they possess basic, neutral, or acid side groups which can be
positively or negatively charged at a given pH.
For an acidic amino acid:

COOH COO-
(CH2)2 (CH2)2
-HN --- C --- CO- <-----> -HN --- C --- CO - Glutamic acid
H H

A <-----> A- + H+
At equilibrium pH = pK = 4.5 k1

k2
k1
k2
 pH Effects
For a basic amino acid:

NH3+ NH2
(CH2)4 (CH2)4
k1 --- C --- CO-
-HN --- C --- CO- <-----> -HN Lysine
H H
k2
Similarly the pK = 10
• So if the active site of an enzyme contains lysine and glutamine,
the enzyme will be most active between 4.5 < pH < 10.
 pH Effects
• a. Each stage of deprotonation
corresponds to a functional group
• (i) First one on left is acid group
• (ii) Second one is amino group
• b. Half-way through titration of
each group is point of inflection
• (i) pH = pKa
• (ii) pKa is measure of tendency to
give-off proton
• (iii) maximum buffer capacity when
pH = pKa
 pH Effects
• pH may alter the 3-D shape of an enzyme
• pH may affect the maximum reaction rate Km
• pH may affect the stability of the enzyme
• pH may affect the affinity of the substrate to the enzyme if the
substrate contains ionic groups.
• Examples - pepsin (stomach) 2< pH < 3.3, amylase (saliva)
optimum 6.8
• Reaction Scheme and Rate Expression - Section 3.3.5.1 in text
Temperature Effects - activation
• Acending part of graph -
temperature activation - rate varies
according to Arrhenius equation:
v = k2 [E]
k2 = Ae-E a/RT
• Ea - activation energy (kcal/mol)
• [E] - enzyme concentration.
• Plot of ln(v) versus 1/T straight line
with slope -Ea/R.
 Temperature Effects -
Inactivation
• Decending part of graph is the temperature
inactivation or thermal denaturation.
•
d[E ]
• [E] = [E0]e -k t
d − = kd [ E ]
dt
• [E 0] initial enzyme concentration
• kd denaturation constant, function of T
kd = Ade-E d/RT
• Ed deactivation energy
• v = Ae-E a/RT [E0]e-k dt
 Energy
• Ea = 4 to 20 kcal/mol
• Ed = 40 to 130 kcal/mol.
• Enzyme denaturation by temperature is
much faster than enzyme activation.
• Increase T from 30 to 40 C, 1.8 fold
increase in enzyme activity but 41 fold
increase in enzyme denaturation.