Lecture 13

Enzyme Inhibi3on and

Control
Reac3on Kine3cs

Bisubstrate reac3ons

Enzyme Inhibi3on

Reversible: Compe33ve, Non-compe33ve, Uncompe33ve

Irreversible (inac3vators)

Control of Enzyma3c Ac3on

1. Allosteric control
2. Mul3ple forms of enzymes
3. Reversible covalent modifica3on
4. Proteoly3c ac3va3on
 Kine3cs of Bisubstrate Reac3ons
60% of known biochemical reac3ons are bisubstrate
Are either transfer or redox reac3on

a. Pep3de
hydrolysis

b. Transfer of hydride
ion from ethanol to
NAD+
Kine3cs of Mul3substrate Reac3ons
E + A + B <-> E + P + Q

1. Sequential Reactions: all substrates must

combine with E before a reaction can occur
a) ordered
b) random
2. Ping-pong Reactions
1. Sequen3al Reac3ons

An Ordered Sequen3al Reac3on

•Required order of S addition

1. Sequen3al Reac3ons
A Random Sequen3al Reac3on
•No preference for the order of S
addition
2. Ping-pong Reac3ons E + A + B <-> E + P + Q

•First product released before second substrate binds

•When E binds A, E changes to F
•When F binds B, F changes back to E
•Group-transfer reac3ons
Double-displacement (Ping-
pong) Reac3on
Bisubstrate Reac3ons Can Be Dis3nguished
by Kine3c Measurements
E + A + B <-> E + P + Q
Sequential Ping-Pong Increasing
Increasing
[B] [B]

1/Vo 1/Vo

1/[A] 1/[A]

Vmax doesn’t change, Both Vmax and Km change

Km changes
 Allosteric Enzymes Do Not Obey
Michaelis–Menten Kine3cs
• Not all enzymes display Michaelis-Menten kine3cs
• An important group of enzymes are allosteric enzymes,
which display coopera3ve substrate binding
• Their kine3cs can o`en be observed as a sigmoidal
reac3on velocity curve
Reac3on Kine3cs

Bisubstrate reac3ons

Enzyme Inhibi3on

Reversible: Compe33ve, Non-compe33ve, Uncompe33ve

Irreversible

Control of Enzyma3c Ac3on

1. Allosteric control
2. Mul3ple forms of enzymes
3. Reversible covalent modifica3on
4. Proteoly3c ac3va3on
Enzymes Can Be Inhibited by Specific
Molecules
• Inhibitor – substance that binds to an enzyme and interferes
with its ac3vity
• Irreversible enzyme inhibitors bind covalently or noncovalently
to the enzyme, but with a negligible dissocia3on constant
• Reversible inhibi3on is characterized by a rapid dissocia3on of
the enzyme-inhibitor complex
Dis3nc3on Between Reversible Inhibitors

There are three common types of

reversible inhibi3on:
1.Compe22ve inhibi2on: The inhibitor is
structurally similar to the substrate and
can bind to the ac3ve site, preven3ng the
actual substrate from binding
2.Uncompe22ve inhibi2on: The inhibitor
binds only to the enzyme-substrate
complex in what is essen3ally substrate-
dependent inhibi3on
3.Noncompe22ve inhibi2on: The
inhibitor binds either the enzyme or
enzyme-substrate complex
 Oseltamivir (Tamiflu®)

• Design of compounds that fit into

an enzyme's ac3ve site is an
essen3al part of modern drug
development
• Tamiflu®-Avian Flu Neuraminidase
Complex
• Neruaminidase hydrolyzes
neruaminic acid (sialic acid) to help
viral par3cles escape from the host
cell
 HIV Enzyme Inhibi3on
HIV par3cles binding HIV injects gene3c material
to a lymphocyte
(RNA) into host cell

HIV Reverse
Transcriptase
RNA DNA

Viral proteis are synthesized as

polyproteins
HIV protease releases viral
proteins by hydrolysis

Compounds to treat AIDS inhibit

these two enzymes
•AZT (azidothymidine) is a nucleoside analog, compete with nucleosides
•AZT is a chain terminator

•The 3' -OH group allows

thymidine to bond to another
nucleo3de via the phosphate
linkage, con3nuing the nucleic
acid chain

•AZT resembles thymidine but has an azido (-N3) group

in the 3' posi3on of the sugar
Glyphosate is a compe33ve inhibitor of the plant enzyme 5-
enoylpyruvylshikimate-3-phosphate synthase

Roundup ready soyben has

been gene3cally modified
with an enzyme that is not
inhibited by glyphosate;
thus round up (glyphosate)
kills the other plants but not
the crop
Compe33ve Inhibi3on: Use of Ethanol to
Treat Methanol Poisoning
ADH

ADH
 Compe22ve Inhibitor (CI)

•CI binds free enzyme and reduces the concentra/on of free

enzyme available for substrate binding
•Competes with substrate for enzyme binding
•Raises Km without effec2ng Vmax
•Can relieve inhibi2on with more S
Compe22ve Enzyme Inhibi3on
Mechanism of a Noncompe22ve Inhibitor

•NI can bind free E or ES complex

•Lowers Vmax
•NIs do not bind to S binding site therefore do not effect Km
•Alters conforma3on of enzyme to effect catalysis but not substrate
binding
Kine3cs of a Noncompe22ve Inhibitor
(NI)
 Uncompe22ve Inhibi3on Involves
Inhibitor Binding to the ES Complex

•UI binds ES complex,

possibly distor3ng the ac3ve
site
•Prevents ES from
proceeding to E + P
•Lowers Km & Vmax
•Adding more substrate
does not reverse the effect
of a UI
Kine3cs of an Uncompe33ve Inhibitor
 Hydrolysis of sucrose to glucose by invertase

14.00

Plots have different 12.00

y = 0.2405x + 3.9216

slopes and y
10.00
intercepts, typical of
1/Vmaxapp
noncompe33ve 8.00

inhibi3on. The same 6.00

intercept on the 4.00

nega3ve y axis, which
gives 1/Km. 1/Km 2.00 y = 0.0976x + 2.1357
1/Vmax
0.00
-30.00 -20.00 -10.00 0.00 10.00 20.00 30.00 40.00
 Irreversible Inhibitors
•Form strong covalent bonds with the enzyme
•May bind at, near, or remote from the ac3ve site
•The basic structure of the enzyme is modified to the degree that it
ceases to work
 Irreversible Inhibitors: Nerve Poisons

•The nerve gases irreversibly

inhibit biological systems by
forming an enzyme-inhibitor
complex with a specific OH group
of serine
•DIFP is a potent neurotoxin
•Inac3vates acetylcholinesterase,
an enzyme that deac3vates the
neurotransmimer acetylcholine
•Causes paralysis, and death
•Used militarily as a nerve gas
Reac3on Kine3cs

Bisubstrate reac3ons

Enzyme Inhibi3on

Reversible: Compe33ve, Non-compe33ve, Uncompe33ve

Irreversible

Control of Enzyma3c Ac3on

1. Allosteric control
2. Mul3ple forms of enzymes
3. Reversible covalent modifica3on
4. Proteoly3c ac3va3on
 Enzyma3c Ac3vity is Regulated in Five
Principal Ways
Enzyme quan3ty – regula3on of gene expression and enzyme
degrada3on (Response 3me = minutes to hours)
a) Transcrip3on
b) Transla3on
c) Enzyme turnover
Enzyme ac3vity (rapid response 3me = frac2on of seconds)
1. Allosteric control
2. Mul3ple forms of enzymes
3. Reversible covalent modifica3on
4. Proteoly3c ac3va3on
 Allosteric Regula3on
a) End products are o`en inhibitors
b) Allosteric modulators bind to site other than the ac3ve site
c) Allosteric enzymes usually have 4o structure
d) Vo vs [S] plots give sigmoidal curve for at least one substrate
e) Can remove allosteric site without effec3ng enzyma3c ac3on
Posi2ve allosteric effects involve
"ac3va3on" of the enzyme - increasing
its ac3vity
Nega2ve allosteric effects involve
"inhibi3on" of the enzyme -
decreasing its ac3vity
 Regula3on of Enzyme Ac3vity
(feedback regula3on)
• 1st commimed step of a biosynthe3c pathway or enzymes at
pathway branch points o`en regulated by feedback
inhibi3on.

4” 5”
H I J
1 A 2 3”
B X C
3’X
5’
E 4’ F G

• Efficient use of biosynthe3c precursors and energy

 Phosphofructokinase (PFK)

Glucose

- +

•PFK catalyzes one of the first steps in

glycolysis
•Phosphoenolpyruvate is an allosteric inhibitor
of PFK
•ADP is an allosteric ac3vator of PFK
Pyruvate
 Allosteric modulators bind to site other
than the ac3ve site and allosteric enzymes
have 4o structure
Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP

ADP

Allosteric Ac2vator (ADP)

binds distal to ac2ve site
Aspartate Transcarbamoylase Is Allosterically
Inhibited by the End Product of Its Pathway

• Aspartate transcarbamoylase
(ATCase) catalyzes the first
step in pyrimidine synthesis
• ATCase is inhibited by the
end product of the pathway,
CTP, in an example of
feedback inhibi3on
• CTP exerts its effects by
binding at a dis3nct
regulatory or allosteric site
on ATCase.
Aspartate Transcarbamoylase Kine3cs
Vo vs [S] plots give sigmoidal curve for at
least one substrate

Binding of substrate
causes structural
changes that convert the
compact, less ac3ve T
state into the expanded,
ac3ve R state.

• Binding of this allosteric inhibitor or this ac3vator does not effect

Vmax, but does alter Km
• Allosteric enzyme do not follow M-M kine3cs
Isozymes Provide a Means of Regula3on
Specific to Dis3nct Tissues and
Developmental Stages
• Isoenzymes or isozymes are enzymes that are
encoded by different genes
• Isozymes catalyze the same reac3on but may display
different regulatory proper3es
• Isozymes may be expressed in a 3ssue-specific or
developmentally specific pamern
• The appearance of certain isozymes in the blood is a
sign of 3ssue damage
Isozymes of Lactate Dehydrogenase
 Covalent modifica3on
•Regula3on by covalent modifica3on is slower than allosteric
regula3on
•Reversible
•Require one enzyme for ac3va3on and one enzyme for
inac3va3on
•Covalent modifica3on stabilizes enzyme T or R conforma3on
•Phosphoryla3on and acetyla3on are common modifica3ons
 Control by Covalent Modifica3on O`en
Involves Protein Phosphoryla3on
•Most common covalent
modifica3on

•Involve protein kinases/

phosphatase

•ATP serves as the phosphate

donor

•Amino acids with –OH groups

are targets for phosphoryla3on

•Phosphates are bulky (-)

charged groups which effect
conforma3on
 Phosphoryla3on is a Highly Effec3ve
Means of Regula3ng the Ac3vi3es of
Target Proteins
• Key features of regula3on by phosphoryla3on :
– The addi3on of the phosphoryl group alters
electrosta3c interac3ons
– A phosphoryl group can form hydrogen bonds
– Phosphoryla3on and dephosphoryla3on can occur
rapidly
– ATP is the cellular energy currency
 Cyclic AMP Ac3vates Protein Kinase A
by Altering the Quaternary Structure
• In muscle cells exposed to epinephrine, cAMP is synthesized

• cAMP s3mulates protein kinase A (PKA) by binding to PKA’s

regulatory (R) subunits, causing their dissocia3on from the
cataly3c subunits. The free cataly3c (C) subunits are in the ac3ve
form.
 Muta3ons in Protein Kinase A
can Cause Cushing’s Syndrome
• Cushing’s syndrome is a collec3on of
diseases resul3ng from excess secre3on
of the hormone cor3sol by the adrenal
cortex
• One cause is now known to be a muta3on
that causes protein kinase A to be
cons3tu3vely ac3ve (i.e., “always on”).
The muta3on causes the cataly3c (C)
subunit to no longer bind the regulatory
(R) subunit so that the enzyme is ac3ve
even when cAMP is absent
• This causes unregulated secre3on of
cor3sol, which has various physiological
effects
 Many Enzymes Are Ac3vated by
Specific Proteoly3c Cleavage
• Proteoly3c cleavage plays a key role in a
number of biochemical processes, for
example:
1. ac3va3on of diges3ve enzymes
2. blood clowng
3. hormone ac3va3on
4. collagen forma3on
5. developmental processes
6. programmed cell death
 Gastric and Pancrea3c Zymogens

• Inactive precursors are also known as zymogens.

 The Genera3on of Trypsin from
Trypsinogen Leads to the Ac3va3on of
Other Zymogens
• Trypsinogen is converted to ac3ve trypsin
by enteropep3dase.
• Trypsin then ac3vates all of the pancrea3c
zymogens.
 Zymogen Ac3va3on by Proteoly3c
Cleavage

• Proteoly3c enzymes are usually synthesized as a larger inac3ve precursor

zymogens, protec3ng the pancreas from self-diges3on
• Enteropep3dase, a serine protease is secreted from the duodenal mucosa
releases the N-terminal pep3de of trypsinogen
• Released trypsin itself is an ac3vator of the reac3on, it is an autocataly3c
reac3on
 Blood Clowng is Accomplished by a
Cascade of Zymogen Ac3va3ons

•Clowng involves series

of zymogen ac3va3ons
•Seven clowng factors
are serine proteases
involved in coagula3on
cascade reac3ons
•Congenital defects in
factor VIII or factor IX
cause abnormal bleeding
or hemophilia.
Forma3on of a Fibrin Clot