Enzyme Kinetics

I.
-

Reaction Kinetics
Rate equations to describe
progress of first-order and
second-order reactions
Michaelis-Menten eqn: Vi Vmax,
Km
Overall catalytic efficiency =
kcat/Km
Lineweaver-Burk plot: present
data and calculate Km, Vmax
Bisubstrate Reactions can be
Ordered/Random/Pingpong
mechanism

Chemical Kinetics described by

Rate Equations
Reaction Order = number of
molecules
participating in Elementary reaction
- reaction velocity
concentration of
reactant (first-order reaction)
- number of molecules that
must collide
to form a product
- 2 molecules needed to form a
product(second-order reaction)
Enzyme Kinetics follow MichaelisMenten Eqn
At S, reaction rate becomes

independent of substrate
concentration (zeroth order)
- k2 becomes rate-limiting step
M-M Equation assumes ES as steady
state
- k-1 >> k1
- Since [S]>>[E], [ES] assumed to
be
constant until [S] depleted
(steady state)

- Michaelis constant, Km = (k-1 +

k1)/k1
- vo = Vmax[S]/(Km + [S])
- Vmax occurs at high [S] or [E] is
saturated
- Km is [S] where reaction rate =
Vmax
- small Km = maximal catalytic
efficiency at
low [S]
kcat/Km as measure of catalytic
efficiency
- kcat = Vmax/[E] number of reaction
processes that each active site
catalyzes/unit
time
- a second order reaction
- when [S] << Km, little [ES] is
formed
- rate of reaction varies directly on
how often
S and E encounter each other
- upper limit: k1
- Rate of decomposition of ES E
+ P not
greater than rate of E + S ES

Kinetic Data provide Vmax and Km

At S, vo Vmax asymptotically

2. Random Mechanism
- no order of preference for
substrate
addition

Nevertheless; steady state fails to

establish the true mechanism
- possibility of other
intermediates
Bisubstrate Reactions
Involve two substrates to form two
products
- oxidation-reduction, or transfer
of
specific functional groups

- both binding sites present in

free
enzyme
Ping Pong reactions
- one of more products released
before all

substrates have been added

- substrates A and B dont
encounter at
the enzyme surface

Sequential Reactions via Single

Displacement
- substrates combine with
enzyme before
reaction may occur = sequential
reaction
1.

Ordered Mechanism

- compulsory substrate addition

to
enzyme
- 1st substrate bind with enzyme

II.
-

to
form binding site for 2nd substrate

Enzyme Inhibition
Inhibitors interact
reversibly/irreversibly to change
Km and/or Vmax
Competitive IN: binds with
active site,
Km
Uncompetitive IN: Km Vmax
Mixed enzyme inhibitor: Vmax,
/Km

Inhibitors
- substances that reduce
enzymes
activity: a) Irreversible - tightly
binds with
enzyme and permanently block
activity
Competitive Inhibition
- directly competes with normal
substrate
at the active site
e.g. malonate inhibits succinate
dehydrogenase
Product inhibition
- products formed may bind at
the
active site, accumulate and
compete
with substrate
- control in cell activity
Transition state analogs
- effective catalysis dependent
on
enzymes ability to bind and
stabilize TS
- compound mimics TS

- INC decreases the free [E] for

substrate
binding
- vo = Vmax[S]/(Km + [S])
- = 1 + [I]/KI
- presence of [I] make [S] smaller

catalytic
nature of the enzyme and not
formation of ES
complex

Mixed (noncompetitive) inhibition

Inhibitor binds to both free E and ES
complex
- metal ions may act as INNC
- if INNC binds to E and ES equally:
only Vmax
affected ; Km unchanged

than it is
- however, at [S], vo Vmax
*Principle behind use of ethanol for
methanol poisoning. Ethanol competes
at active site, versus methanol
slowing production of formaldehyde
methanol excreted through urine
Uncompetitive Inhibition
Inhibitor binds directly to ES complex
but not
to free enzyme
- distorts the active site
inactivated enzyme
- addition of substrate wont affect
the effect
- substrate binding doesnt affect
binding of inhibitor
- require that inhibitor affect the

Irreversible Inhibitor
- resemble pure noncompetitive
inhibitor =

decreasing free [E] at all [S]

III. Control of Free Enzyme

- Allosteric effectors bind with
multisubunit enzymes
cooperative conformation
change catalytic activity
- Phosphorylation
Dephosphorylation

induce control by shifting to

more/less active conformation

cooperatively

Enzyme must be able to control and

coordinate its activity depending on its
metabolic need

Ways to control:
1.

Enzyme Availability

- amount of enzyme depend on

its
synthesis degradation
- controlled by cell and dramatic
changes over time
2.

Enzyme Activity

- modulation of activity by
structural
alteration that influence ES
complex
formation or turnover number
- allosteric effectors that cause
changes
in enzymatic activity
- covalent modification, usually
phodephosphorylation of specific Ser,
Thr, Tyr
residues
Allosteric Control by binding at
site not the active site
e.g. aspartate carbamoylase
(ATCase)
- catalyzes N-carbamoyl
aspartate
carbamoyl phosphate +
aspartate
- both substrate bind

- allosterically inhibited by
cytidine
triphosphate (CTP), pyrimidine
nucleotide
- allosterically activated by
adenosine
triphosphate (ATP), purine
nucleotide
- CTP, a feedback inhibitor,
inhibits its
earlier step in its biosynthesis
- [CTP], CTP synthesis
- [CTP], CTP dissociates from
ATCase
CTP synthesis
- coordination of ATP and CTP in
purine
and pyrimidine synthesis
- [ATP]>[CTP] ATCase
activated to
form pyrimidine nucleotides until
equal
- [CTP]>[ATP] permits ATP
synthesis

to balance out pyrimidine-purine

conc.
Allosteric effectors lead to structural
changes
- Both CTP and ATP bind to same site
- CTP: T-state binding and stability
- ATP: R-state binding and stability
- CTP + R-state contraction of
regulatory
dimer (similar to T-state, less active)

reorientation of active site

decrease in
activity
- ATP + T-state trimer move apart
(like Rstate) increase in activity
Control by Covalent Modification
(Phosphorylation)
- processes catalyzed by protein
kinases and
protein phosphatases
e.g. glycogen phosphorylase
- catalyzes phosphorolysis
(bond
cleavage + phosphate group
substitution)
of glycogen to yield glucose-1phosphate
(G1P)
- rate controlling step in
glycogen
breakdown for fuel in metabolic
processes
Phosphorylation-dephosphorylation
resemble
allosteric control
Glycogen phosphorylase
- T state: inactive = malformed
active site
and surface loop (residues 282284)

blocking active site from

substrate
- R state: active = Arg 569
oriented to
allow binding with substrate and
282-284
no longer blocks active site
Phosphorylation of Ser 14 promotes
T(inactive)R(active)
conformational change
phosphorylase a (pa) =

phosphorylated form
+
phosphoryl group
esterified to Ser 14
- glucose as only allosteric
effector
- binds to T-state inactivating
enzyme
phosphorylase b (pb) =
dephosphorylated form
- ATP, glucose-6-phosphate
(G6P, upon
conversion of G1P) prefers
binding with T
state of pb inactivating enzyme
- AMP prefers binding with R state
of pa
activating enzyme
muscle excertion; ATP, G6P,
glucose
concentration (thus no need for
glycogen
breakdown)
muscle excertion; AMP (induce
breakdown to help fuel metabolism)
In phosphorylase a,

- Ser 14 phosphate group forms ion

pairs
with two cationic Arg side chains
linking
active site, subunit interface, N-

terminal
region glycogen phosphorylases
helices
pull apart triggers quaternary T to
R
transition
* Ser 14 acts as internal allosteric

effector to
shift T R equilibrium toward R
state
Cascades of Protein Kinases = Greater
sensitivity
- protein kinases/phosphatases
linked in
series
- greater capacity at signal
amplification
because small fractional change in
effector
concentration cause larger fractional
enzymatic activity