B BC Chap13

NTOU 2011
NTOU 2010
Enzyme
y
Kinetics
Reginald H. Garrett
Charles M. Grisham
Lins B
Biochemistryy Lecture
Lins B
Chapter 13
Essential Questions
Before this class, ask your self the following
questions:
What are enzymes?

How do enzymes work?
How many enzymes you know?
What is kinetics?
What is enzyme inhibitor?
iuuq;00mnt/mt/oupv/fev/ux0dpvstf0217
ibokjbAnbjm/oupv/fev/ux
2
NTOU 2010
NTOU 2010
Lins B
Lins B
Outline
Part I: Get to know enzymes
Enzyme
y
Features
Enzyme nomenclature
Activity
Unusual enzymes..
Part II: Enzyme kinetics

Part III: Inhibitor and bimolecular kinetics
Virtually All Reactions in Cells Are

Mediated by Enzymes
Enzymes catalyze thermodynamically favorable
reactions causing them to proceed at
reactions,
extraordinarily rapid rates (see Figure 13.1)
Enzymes
En mes provide
pro ide cells with
ith the abilit
ability to e
exert
ert
kinetic control over thermodynamic potentiality
Living systems use enzymes to accelerate and
control the rates of vitally important biochemical
reactions
Enzymes are the agents of metabolic function
4
NTOU 2010
NTOU 2010
Lins B
Lins Biochemistryy Lecture
Figure
gu e 13.1
3 Reaction
eact o p
profile
o e sshowing
o
g tthe
e large
a ge free
ee
energy of activation for glucose oxidation. Enzymes lower
G, thereby accelerating rate.
13.1 What Characteristic Features Define

Enzymes?
Catalytic
y power
p
Specificity
R
Regulation
l ti
y
nomenclature
Enzyme
Coenzymes and cofactors
NTOU 2010
NTOU 2010
Specificity
Lins B
Lins B
Catalytic Power
Catalytic power is defined as the ratio of the
enzyme-catalyzed
t l
d rate
t off a reaction
ti to
t the
th
uncatalyzed rate
Enzymes can accelerate reactions as much
as 1016 over uncatalyzed rates
Urease is a g
good example:
p
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10 -10/sec
Ratio is 1x1014
Enzymes selectively recognize proper

substrates
subs
a es o
over
e o
other
e molecules
o ecu es
Enzymes produce products in very high
yields - often much greater than 95%
Specificity is controlled by structure - the
unique fit of substrate with enzyme controls
the selectivity for substrate and the product
yield
NTOU 2010
NTOU 2010
Regulation
90% yield in each step; 35% over 10 steps

Lins B
Lins B
Figure 13.3 Yields in biological

reactions must be substantially
greater than 90%.
Enzymes are the Agents of

Metabolic Function
Regulation of enzyme
activity ensures that the
rate of metabolic reactions
is appropriate to cellular
requirements
Figure 13.2 The breakdown of glucose
by glycolysis provides a prime example
of a metabolic pathway.
10
NTOU 2010
NTOU 2010
Lins B
Lins B
Enzyme nomenclature
Common name:
Suffix ase to substrate
Urease: urea hydrolyzing enzyme
Phosphatase: hydrolyzing phosphoryl group
Enzyme
y
Nomenclature Provides a Systematic
y
Way of Naming Metabolic Reactions
What is the activity of following enzymes?

Catalase
Trypsin
Pepsin
Systematic classification
6 major classes of reaction
11
12
NTOU 2010
NTOU 2010
Coenzymes
y
and Cofactors
Lins B
Lins B
Example:
Classification of the enzyme catalyzing the
following reaction:
ATP + D-glucose ADP + D-glucose-6phosphate
Phosphate group transferred
Nonprotein components essential to enzyme

activity
Cofactor = vitamin?
Prosthetic group?
Holoenzyme
y
vs. apoenzyme
p
y
Transferase
T
f
((class
l
2)
transferring P-containing group (subclass 7)
With an alcohol group as acceptor (sub-subclass 1)
Entry 2: glucokinase (E.C.2.7.1.2.)
Entry 1: hexokinase (E
(E.C.2.7.1.1)
C 2 7 1 1)
What is kinase?
13
14
NTOU 2010
NTOU 2010
Why
y Enzymes
y
Are So Specific?
p
Lins B
Lins B
The Lock and key hypothesis

The Induced fit hypothesis
Induced fit favors formation of the transition
transitionstate
Specificity and reactivity are often linked.
C t l i off Hexokinase
Catalysis
H
ki
hexokinase example
binding of glucose in the active site induces a
conformational change in the enzyme
causes the two domains of hexokinase to close around
the substrate, creating the catalytic site
15
16
NTOU 2010
NTOU 2010
Enzymatic Activity and pH

Lins B
Lins B
Enzyme-substrate recognition and catalysis

are greatly dependent on pH
Enzymes have a variety of ionizable side
chains that determine its secondary
y and
tertiary structure and also affect events in the
active site
Substrate may also have ionizable groups
Enzymes are usually active only over a limited
range of pH
Enzymatic
E
ti Activity
A ti it is
i St
Strongly
l
Influenced by pH
Figure 13
13.11
11 The pH activity profiles of four different enzymes
enzymes.
17
18
NTOU 2010
NTOU 2010
Lins B
Lins B
The Response of Enzymatic

Activity to Temperature
Rates of enzyme-catalyzed reactions generally
increase with increasing
g temperature
p
However, at temperatures above 50 to 60 C,
enzymes typically show a decline in activity
Two effects here:
Enzyme
E
rate
t typically
t i ll d
doubles
bl iin rate
t ffor ever
10C as long as the enzyme is stable and
active
ti
At higher temperatures, the protein becomes
unstable and denaturation occurs
19
The effect of temperature on enzyme activity
20
NTOU 2010
NTOU 2010
Lins B
Lins B
Unusual Enzymes
Rib
Ribozymes - segments
t off RNA th
thatt di
display
l
enzyme activity in the absence of protein
Examples:
E
l
RN
RNase P and
d peptidyl
id l
transferase
Abzymes
y
- antibodies raised to bind the
transition state of a reaction of interest
For a good review of abzymes, see Science,
Vol. 269, pages 1835-1842 (1995)
Transition states are covered in more depth
in Chapter 14
RNA Molecules That Are Catalytic Have

Been Termed Ribozymes
Figure 13.25 RNA splicing in

Tetrahymena rRNA maturation
maturation.
21
22
NTOU 2010
NTOU 2010
Lins B
Lins B
RNA Molecules That Are Catalytic Have

RNA Molecules That Are Catalytic

y Have
Figure 13
13.26
26 (a) The 50S subunit from H.
H marismortui
marismortui. (b) The
aminoacyl-tRNA (yellow) and the peptidyl-tRNA (orange) in the
peptidyl
p
p y transferase active site.
Figure 13.27 The peptidyl transferase reaction.
23
24
NTOU 2010
NTOU 2010
Figure 13
13.28
28 (a)
Intramolecular
hydrolysis of a
hydroxy ester yields
a -lactone.
Lins B
Lins B
Antibody
y Molecules Can Have Catalytic
y
Activity
13.8 Is It Possible to Design An Enzyme to

Catalyze Any Desired Reaction?
A known enzyme can be engineered
engineered by in vitro
mutagenesis, replacing active site residues with
new ones that might catalyze a desired reaction
Another approach attempts to design a totally
new protein with the desired structure and
activity
This latter approach often begins with studies in
in
silico i.e., computer modeling
Protein folding and stability issues make this
approach more difficult
And the cellular environment may provide
complications not apparent in the computer modeling
(b) The cyclic

phosphonate ester
analog of the cyclic
transition state.
25
26
NTOU 2010
NTOU 2010
Lins B
Lins B
13 8 Is It Possible to Design An Enzyme to

13.8
Catalyze Any Desired Reaction?
Figure 13
13.29
29 cis
cis-1
1,2
2-Dichloroethylene
Dichloroethylene (DCE) is an
industrial solvent that poses hazards to human health.
Site-directed mutations have enabled the conversion of a
bacterial epoxide hydrolase to catalyze the chlorinated
epoxide
id h
hydrolase
d l
reaction.
ti
27
End of Part 1
Ask yourself
What are g
general features of an enzyme?
y
Why and how an enzyme could be so
specific?
Are all enzymes proteins?
What
Wh t kind
ki d off enzyme can you b
buy and
d use iin
your daily life? Where are they come from?
Can we design an enzyme by ourselves?
28
NTOU 2010
NTOU 2010
Lins B
Lins B
13.2 Can the Rate of an Enzyme-Catalyzed

y
y
Reaction Be Defined in a Mathematical Way?
What is Kinetics?
the branch of science concerned with the rates of
reactions
ti
What can we learn from enzyme kinetics?
to
t determine
d t
i the
th maximum
i
reaction
ti velocity
l it and
d
binding affinities for substrates and inhibitors
Why we have to learn about enzyme kinetics?
To know insights of enzyme mechanisms and
metabolic pathways
This information can be exploited to control and
manipulate the course of metabolic events
(Pharmaceutical purpose)
Several kinetics terms to understand
rate or velocity
rate constant
rate law
order of a reaction
molecularity of a reaction
29
30
NTOU 2010
NTOU 2010
Before we learn enzyme kinetics

kinetics
Lins B
Lins B
Lets review some general ideas

Chemical kinetics
Activation energy
Transition state
Reaction rate
Chemical Kinetics Provides a Foundation

for Exploring Enzyme Kinetics
Enzyme kinetics is based on chemical
kinetics:
First-order reaction = unimolecular
reaction molecularity =1
The simple elementary reaction of AP
A P is a
first-order reaction
Examples
Intramolecular rearrangement reaction
Isotope decay
31
32
NTOU 2010
NTOU 2010
The Time-Course
Time Course of a First
First-Order
Order Reaction
Lins B
Lins B
Chemical Kinetics: First-order

First order reaction
Consider a reaction of overall stoichiometry as
shown:
Ao P
d [ P]
v
dt
[[ A]
v
dt
d [ A]
dt
k[ A]
Figure 13.4 Plot of the course of a first-order reaction. The

h lf ti
half-time,
t1/2 is
i th
the titime ffor one-half
h lf off th
the starting
t ti amountt off
A to disappear.
The rate is proportional to the concentration of A

33
NTOU 2010
34
NTOU 2010
Chemical Kinetics: Second-order

Second order reaction
Summary of Chemical Kinetics

Lins B
Lins B
Bimolecular reaction = second-order reaction

Molecularities = 2
A+BP+Q
the rate law is
v = k [A][B]
2AP+Q
v = k [A]2
Molecularities > 2 is rare and never

greater 3!
g
R
Remember:
b Ki
Kinetics
ti cannott prove a
reaction mechanism. They can only rule
out various alternative hypotheses!
Question: What is the unit of rate constant (k)?

35
36
NTOU 2010
NTOU 2010
Two ways to increase chemical reaction rate!

Lins B
Lins B
Energy diagram for a chemical reaction (AP)
Transition state
The transition state sits at the apex of the
energy profile
fil iin th
the energy di
diagram
A typical enzyme-catalyzed reaction must pass
through a transition state
proportional
p
to the
The reaction rate is p
concentration of reactant molecules with the
transition-state energy
gy
(a) raising the temperature from T1 to T2

(b) adding a catalyst.
37
38
NTOU 2010
NTOU 2010
Activation energy
Lins B
Lins B
Free energy of activation, 'G

the energy required to raise the average energy of
1 mol of reactant to the transition state energy (at a
given temperature).
Decreasing G increases the reaction rate

gy is related to the rate
The activation energy
constant by Arrhenius equation:
13 3 What Equations Define the Kinetics of

13.3
Enzyme-Catalyzed Reactions?
Enzyme is not involved in the chemical kinetics
of the reaction!
True or False?
A simple first-order reactions

display a plot of the reaction rate vs. reactant
concentration as a straight line (Figure 13.6)
If the same reaction with an enzyme involved, is
anything changed?
Ae ''G / RT
Note the difference between 'G and 'G

39
40
NTOU 2010
NTOU 2010
Enzyme did change the chemical kinetics

Lins B
Lins Biochemistryy Lecture
Truth is more complicated!

At low concentrations of the substrate
The rate is proportional to S
S, as in a first-order
first order
reaction
At higher
hi h concentrations
t ti
off substrate
b t t
The enzyme reaction approaches zero-order
kinetics
Figure 13.6
A plot of v vs. [A] for the unimolecular chemical reaction,
A P yields a straight line having a slope equal to kk.
AP,
41
42
NTOU 2010
NTOU 2010
Lins B
Lins B
Saturation Effect
We need a new equation for enzymecatalyzed chemical kinetics!

Louis Michaelis and Maud Menten
invented a new equation which became
q
of enzyme
y
the fundamental equation
kinetics.
What can we learn from M
M-M
M equation?
We could know two major indexes of the
enzyme: Km and Vmax!
As [[S]] increases,, kinetic behavior changes

g from
st
1 order to zero-order kinetics
43
44
NTOU 2010
NTOU 2010
Two More Assumptions of M

M-M
M equation
Lins B
Lins B
Th Mi
The
Michaelis-Menten
h li M t Equation
E
ti
A
Assumptions
ti
off M-M
M M enzyme reaction
ti
1. It assumes the formation of an enzymesubstrate complex
2. It assumes that the ES complex is in
rapid equilibrium with free enzyme
3. Breakdown of ES to form products is
assumed to be slower than
1) formation of ES and
2) breakdown of ES to re
re-form
form E and S
4. [ES] Remains Constant:

also known as steady state
assumption
ti by
b Briggs
Bi
&
Haldane in 1925
5 Rate measurement is
5.
finished right after
Substrate added
D[ES]/dt = 0
To ignore E + P ES
These assumption would

simplify the following
calculating
45
46
NTOU 2010
NTOU 2010
Lins B
Lins B
Understanding Km
Km is
i th
the "kinetic
"ki ti activator
ti t constant
t t derived
d i d
from rate constants
Learning Point!
Three meanings of Km!
How to get Km of an enzyme?
Km Step 1
According to M-M assumption, enzyme
presented as
reaction could be p
The
e sy
synthesis
t es s rate
ate o
of ES
S
Vf = k1 [E] [S]
Therefore, the dissociation of ES
Vd = k-11 [ES] + k2 [ES]
Please pay attention!
47
48
NTOU 2010
NTOU 2010
Lins B
Lins B
Km Step 2
Briggs & Haldane assumed that Vf = Vd
under steady-state!
y
Therefore.
Vf = k1 [E] [S] = k-1 [ES] + k2 [ES] =V
Vd (Eq1)
(E 1)
Please
ease remember
e e be
[E]: concentration of enzyme without substrate
binding!
g
[ES]: concentration of enzyme binding with substrate!
We dont know [[E]] and [ES],
[ ], actually.
y We onlyy know
[ET]: concentration of total enzyme!
Km Step 3
Because [ET] = [E] + [ES]
Therefore [E] = [ET] - [ES]
Try to reduce unknown value in Eq1
k1 [E] [S] = k-1 [ES] + k2 [ES]
k1([ET] - [ES]) [S] = (k-1 + k2) [ES]
Move all constants to one side, then.
([ET] - [ES]) [S]
k-1 + k2
k1
[ES]
Km (Eq2)
49
50
NTOU 2010
NTOU 2010
Km
k-11 + k2
k1
Lins B
Lins B
The First Meaning of Km

Sum of ES dissociation rate constants
ES synthesis rate constant
An enzyme with bigger Km:

Dissociation rate constants are larger or
synthesis rate constant is smaller
Literally,
Li
ll substrate
b
and
d enzyme are diffi
difficult
l to
bind!
What if an enzyme with small Km?

Therefore,
Therefore Km is related to the enzymeenzyme
substrate binding capacity!
Km Step 4
In all kinds of kinetics studies, the most
p
issue is reaction rate,, V !
important
V = k2 [ES]
(Eq3)
We
W dont
d t kknow [ES] exactly,
tl but
b t we
know
Km
( T] - [ES])
([E
S ) [S]
S
[[ES]]
Tryy to move [[ES]] to one side

51
52
NTOU 2010
NTOU 2010
Lins B
Lins B
The Second Meaning of Km

After [ES] moved to one side
side, Eq2
becomes..
[ET] y [S]
[ES]
(Eq4)
Km + [S]
Now combine Eq3 and Eq4, you will get

V
k2 [ET] y [S]
(Eq5)
Km + [S]
k2 [ET] y [S]
(Eq5)
Km + [S]
As you see in Eq5, V increases when [S]

increased.
increased
If [S] becomes very high, what happened
t V?
to
Enzyme
y
will be saturated!
Km + [S] [S]
V Vmax
Km is related to the reaction rate!

An enzyme with smaller Km may has higher reaction
rate!
Km Step 5
53
54
NTOU 2010
NTOU 2010
Lins B
Lins B
Km Step 6
Now
N
E
Eq5
5b
becomes
k2 [ET] y [S]
Vmax
[S]
k2 [ET]
(Eq6)
We could also combine Eq5 and Eq6,

p
make it simpler!
V
Vmaxy [[S]]
Km + [S]
The Third Meaning of Km

From Eq7, we could redefine the meaning
of Km as..
Km
[S]y(Vmax V)
V
That
at means
ea s when
e
Vmax
V
[S]y((
[S]
Vmax
1) (Eq8)
V
- 1 = 1,, Km = [S]
V
Also means max = 2 V = Vmax
V
(Eq7)
55
When reaction rate is Vmax, the

corresponding [S] equals to Km!
56
NTOU 2010
NTOU 2010
Lins B
Lins B
Take a look at the Km values for some

enzymes and their substrates
O h iindexes
Other
d
ffor an enzyme
kcat, the turnover number
A measure of catalytic activity
Defines the activity
y of one molecule of enzyme
y
If the M-M model fits, k2 = kcat = Vmax/Et
Values of kcat range from less than 1/sec to
many millions per sec
57
58
NTOU 2010
NTOU 2010
Lins B
Lins B
The Turnover Number Defines the Activity

of One Enzyme Molecule
O h iindexes
Other
d
ffor an enzyme
The Ratio kcat/Km
Defines the catalytic efficiency of an
enzyme
An estimate of "how perfect" the enzyme is
kcat/Km is an apparent
pp
second-order rate
constant
It measures how the enzyme performs when S
i llow
is
The upper limit for kcat/Km is the diffusion limit the rate at which E and S diffuse together
59
60
NTOU 2010
NTOU 2010
Lins B
Lins B
The Ratio kcat/Km Defines the Catalytic

y
Efficiency of an Enzyme
How to measure the Km?

The nature of Michaelis-Menten
Michaelis Menten equation:
Combination of 0-order and 1st-order kinetics
Describes
D
ib a rectangular
t
l h
hyperbolic
b li
dependence of v on S
According to Eq8
Vmax
But this is not good for

calculating
calculating
[S] here is Km
Catalytic perfection
61
62
NTOU 2010
NTOU 2010
Lins B
Lins B
Because of Vmax
Vmax is a theoretical maximal velocity and is
a constant
Vmax is NEVER achieved in reality
For
F example:
l
To reach a rate of 99% Vmax
V
Vmax
0.99
Linear Plots Can Be Derived from the

Michaelis-Menten Equation
V
That means you have to prepare

substrate [S] = 99 Km
It is not possible practically.
63
Km + [S]
(Eq7)
Rearrange to obtain the Lineweaver-Burk

equation:
ti
1
v
[S]
Km + [[S]]
Vmaxy [S]
Km
Vmax
1
1

[
S
]
Vmax
A plot
l t off 1/
1/v versus 1/[S] should
h ld yield
i ld a straight
t i ht
line
64
NTOU 2010
NTOU 2010
The Lineweaver-Burk double-reciprocal plot

Lins B
Lins B
Y = aX + b
H
Hanes-Woolf
W lf Linear
Li
Plot
Pl t
Begin with Lineweaver
Lineweaver-Burk
Burk and divide both
sides by [S] to obtain:
[S ]
v
Vmax
Km
[
S
]

Vmax
65
66
NTOU 2010
NTOU 2010
Hanes Woolf Plot is Better - Why?

Hanes-Woolf
Lins B
Lins B
Hanes-Woolf plot
p
[S]/v versus [S]
smaller and more
consistent
i t t errors across the
th
plot
More about kinetics

pH may effect on Km or Vmax or both in
y
kinetics
enzyme
What if the plot is not linear?
Regulatory enzymes (allosteric enzymes)
67
68
NTOU 2010
NTOU 2010
Lins B
Lins B
End of Part 2
Ask yourself
How many
y indexes you
y have learned to
describe the kinetic properties of an enzyme?
What is M
M-M
M equation? Do you know whats
what s
different from enzyme kinetics and chemical
kinetics?
Do you understand the three meanings of Km
and know how to conduct from Eq1 to Eq8?
Do you understand how to plot LineweaverB k plot,
Burk
l t and
d estimate
ti t Km and
d Vmax from
f
it?
13.4 What Can Be Learned from the

Inhibition of Enzyme Activity?
Enzymes may be inhibited reversibly or
irreversibly
Reversible inhibitors
may bind at the active site or at some other site
Competitive inhibition
Noncompetitive inhibition
Uncompetitive inhibition
Irreversible inhibitors
Covalent
C
l t modification
difi ti
Kinetically similar to ___cometitive inhibition!
69
NTOU 2010
70
NTOU 2010
Lins B
Lins B
Irreversible inhibitor
Example:
Penicillin is an irreversible
inhibitor of glycopeptide
transpeptidase
Reversible Inhibitors May Bind at the

Active S
Site or at S
Some O
Other S
Site
Suicide inhibitor
catalyzes
t l
an essential
ti l step
t
in bacterial cell all synthesis
71
72
NTOU 2010
NTOU 2010
Lins B
Lins B
Competitive inhibition
Inhibitor, I, also binding to enzyme and
p
for the same binding
g site with
compete
substrate
E + S
E + I
K1
K-1
K3
K-3
K2
ES
Kinetic of competitive inhibition

Step 1
Because [[ET] = [E]
[ ] + [ES]
[ ] and
([ET] - [ES]) [S]
E + P
k1
[ES]
EI
So
k-1 + k2
[ES] =
Km (Eq2)
[E] [S]
Km
(Eq9)
73
74
NTOU 2010
NTOU 2010
Because
E + I
K3
K-3
EI
Vf = Vd under steady-state!
Therefore.
Vf = k3 [E] [I] = k-3 [EI]
[EI] =
k3
k-3
Lins B
Lins B
Kinetic of competitive
p
inhibition
Step 2
[E] [I] = 1/kI [E] [I]
(Eq10)

Step 3
Knowing [ET] = [E] + [ES] + [EI]
[ET] = [E] +
[E] [S]
[E] [I]
+
Km
KI
(Eq12)
Put [E] to one side

side.
(E 11)
(Eq11)
[E] =
75
KI Km [ET]
(KI Km + KI [S] + Km [I])
(Eq13)
76
NTOU 2010
NTOU 2010
Lins B
Lins B

Step 4
Rate is still the most important!
V = k2 [ES]
According to Eq9
[ES] =
[E] [S]
Km
it could be changed to
to
k2 [[E]] [S]
[ ]
V=
Km
(Eq9)
Combine Eq13 and Eq14 to redefine rate!

V =
k2 KI Km [ET] [S]
Km(KI Km + KI [S] + Km [I])
(Eq15)
Because Vmax = k2 [ET]
(Eq14)
Vmaxy [S]
[S] + Km ( 1 + [I]/KI)
(Eq16)
77
78
NTOU 2010
NTOU 2010
Lins B
Lins B
Effect of Competitive Inhibitors on

Lineweaver-Burk plot
V

Step 5
Vmaxy [S]
[S] + Km ( 1 + [I]/KI)
M
[I]
[I],
More
Less rate!
Larger Km!
Why?
Example of Competitive Inhibition

Succinate Dehydrogenase (SDH)
Vmax unchanged!
Why?
y
79
80
NTOU 2010
NTOU 2010
S and I bind to different sites on the enzyme

I could bind to E or ES
2 KI
2 types:
Lins B
Lins B
Noncompetitive inhibition
ES + I
KI
KI
Inhibitor binds to E or ES equally

KI = KI
Km not change!
Why?
Wh ?
Vmax smaller!
Pure noncompetition
Mixed noncompetition
E + I
Pure Noncompetitive Inhibition
Why?
EI
IES
81
82
NTOU 2010
NTOU 2010
Lins B
Lins B
Mixed Noncompetitive Inhibition

Binding of I influences binding of S
2 situations:
Uncompetitive Inhibition
I combines only with ES and affect k2
(kcat)!
Km smaller!
Why?
Wh ?
Vmax smaller!
Why?
Slope is not changed!

Why?
83
84
NTOU 2010
NTOU 2010
Lins B
Lins B
Bimolecular Reactions Catalyzed

by Enzymes
Most enzymes catalyze reactions involving
two (or more) substrates
2 Reaction mechanisms
Sequential (single-displacement)
reactions
Ping-pong (double-displacement)
reactions
ti
Single displacement Reactions

Single-displacement
Two distinct classes:
Random: either substrate may
y bind first,,
followed by the other substrate
Ordered: a leading substrate binds first,
first
followed by the other substrate
Similar
Si il tto noncompetitive
titi iinhibition!
hibiti !
Why?
85
86
NTOU 2010
NTOU 2010
All possible binary enzyme-substrate and enzymeproduct complexes are formed rapidly and
reversibly
Conversion of AEB to PEQ is the Rate-Limiting Step
If A has no influence on B binding purely random!
87
Lins B
Lins B
Random Single
Random,
Single-Displacement
Displacement Reactions
Example of Random, Singledisplacement enzyme

Creatine Kinase (important in muscle)
+ ATP
Creatine
Kinase
+ ADP
88
NTOU 2010
NTOU 2010
Lins B
Lins B
Lineweaver Burk Plot for single

Lineweaver-Burk
singledisplacement bisubstrate enzyme
Purely random
Ordered Single
Ordered,
Single-Displacement
Displacement Reaction
The leading substrate (obligatory or
compulsory substrate), A, must bind first
followed by B.
Reaction
R
ti b
between
t
A and
d B occurs iin th
the
ternary complex and is usually followed by
an ordered release of the products, P and Q.
89
90
NTOU 2010
NTOU 2010
Lins B
Lins B
An Alternative way of Portraying the

Ordered, Single-Displacement Reaction
A and P are competitive for enzyme!
A and B are not competitve for enzyme.
Example for Ordered, SingleDisplacement Reaction

Alcohol dehydrogenase (ADH)
Use NAD+ ((nicotinamide adenine dinucleotide))
as coenzyme or compulsory substrate
NAD+ + CH3CH2OH
No A
A, no B binding!
(A)
91
(B)
NADH + H+ + CH3CHO
(Q)
(P)
92
NTOU 2010
NTOU 2010
Lins B
Lins B
Double Displacement Reaction

Aka. Ping-Pong reactions
Formation of a covalently
y modified enzyme
y
intermediate.
The product of the enzymes
enzyme s reaction with A
(called P in the above scheme) is released
prior to reaction of the enzyme with the
second substrate, B.
An Alternative Presentation of the DoubleDisplacement (Ping-Pong) Reaction

A and Q compete for E
E
P and B compete for E
93
94
NTOU 2010
NTOU 2010
Lins B
Lins B
Lineweaver-Burk
Lineweaver
Burk Plot for the Double
Displacement Reaction
Similar to
uncompetitive
i hibiti
inhibition,
why?
h ?
95
Example for the Double Displacement

Reaction
Glutamate:aspartate
Aminotransferase
Amino acid
metabolism
Enzyme bond
coenzyme
Pyridoxal
y
p
phosphate
p
96
NTOU 2010
NTOU 2010
Lins B
Lins B
End of Part 3
Ask yourself
What is the difference of reversible and
irreversible inhibition?
What is competitive,
competitive noncompetitive
noncompetitive,
uncompetitve inhibition? What are they look
like in Lineweaver-Burk
Lineweaver Burk plot?
End of this chapter

You should know
97
What characteristic features define enzymes?

How can enzymes be so specific?
Are all enzymes proteins?
Is it possible to design an enzyme to catalyze any desired
reaction?
Can the rate of an enzyme-catalyzed reaction be defined in a
mathematical way?
What equations define the kinetics of enzyme-catalyzed
reactions?
What can be learned from the inhibition of enzyme activity?
What is the kinetic behavior of enzymes catalyzing bimolecular
reactions?
98

B BC Chap13

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

B BC Chap13

Uploaded by

Copyright:

Available Formats

NTOU 2011

What are enzymes?

Part II: Enzyme kinetics

Virtually All Reactions in Cells Are

Lins Biochemistryy Lecture

13.1 What Characteristic Features Define

Enzymes selectively recognize proper

90% yield in each step; 35% over 10 steps

Figure 13.3 Yields in biological

Enzymes are the Agents of

What is the activity of following enzymes?

Nonprotein components essential to enzyme

The Lock and key hypothesis

Enzymatic Activity and pH

Enzyme-substrate recognition and catalysis

The Response of Enzymatic

The effect of temperature on enzyme activity

RNA Molecules That Are Catalytic Have

Figure 13.25 RNA splicing in

RNA Molecules That Are Catalytic Have

RNA Molecules That Are Catalytic

13.8 Is It Possible to Design An Enzyme to

(b) The cyclic

13 8 Is It Possible to Design An Enzyme to

13.2 Can the Rate of an Enzyme-Catalyzed

Several kinetics terms to understand

Before we learn enzyme kinetics

Lets review some general ideas

Chemical Kinetics Provides a Foundation

Chemical Kinetics: First-order

Figure 13.4 Plot of the course of a first-order reaction. The

The rate is proportional to the concentration of A

Chemical Kinetics: Second-order

Summary of Chemical Kinetics

Bimolecular reaction = second-order reaction

Molecularities > 2 is rare and never

Question: What is the unit of rate constant (k)?

Two ways to increase chemical reaction rate!

Energy diagram for a chemical reaction (AP)

(a) raising the temperature from T1 to T2

Free energy of activation, 'G

Decreasing G increases the reaction rate

13 3 What Equations Define the Kinetics of

A simple first-order reactions

Note the difference between 'G and 'G

Enzyme did change the chemical kinetics

Lins Biochemistryy Lecture

Truth is more complicated!

We need a new equation for enzymecatalyzed chemical kinetics!

As [[S]] increases,, kinetic behavior changes

Two More Assumptions of M

4. [ES] Remains Constant:

These assumption would

Please pay attention!

The First Meaning of Km

ES synthesis rate constant

An enzyme with bigger Km:

What if an enzyme with small Km?

Tryy to move [[ES]] to one side

The Second Meaning of Km

Now combine Eq3 and Eq4, you will get

As you see in Eq5, V increases when [S]

Km is related to the reaction rate!