Chapter 10
Regulatory strategies
Like motor traffic, metabolic pathways
flow more efficiently when regulated by
signals.
Enzymatic activity is regulated
in five principal ways :
1. Allosteric control. Allosteric proteins contain distinct regulatory sites and
multiple functional sites. cooperativity
2. Multiple forms of enzymes. Isozymes, or isoenzymes, provide an avenue
for varying regulation of the same reaction at distinct locations or times.
3. Reversible covalent modification. The catalytic properties of enzymes are
altered by the covalent attachment of a modifying group. Kinase - phosphatase
4. Proteolytic activation : Irreversible convert an inactive enzyme into an
active one.
5. Controlling the amount of enzyme present. Transcription level
10.1 Aspartate transcarbamoylase is allosterically
inhibited by the end product of its pathway
Aspartate transcarbamoylase catalyzes the first step in the
biosynthesis of pyrimidines(such as cytidine triphosphate CTP).
ATCase is inhibited by CTP, the
final product of the ATCaseinitiated pathway.
-The inhibition of ATCase by CTP is an example of feedback
inhibition, the inhibition of an enzyme by the end product of the
pathway.
-CTP is structurally quite different from the substrate and product of
the reaction.
-Thus CTP must bind to a site distinct from the active site (allosteric or
regulatory site).
Allosterically regulated enzymes do not follow MichaelisMenten kinetics
-The curve differs from that
expected for an enzyme that
follows Michaelis-Menten
kinetics.
-Sigmoidal kinetics
-In hemoglobin, sigmoidal
curve from cooperativity
ATCase consists of separable catalytic and regulatory
subunit
-ATCase can be separated into regulatory(r) and catalytic(c) subunits
; how to know? evidence?
-p-hydroxymercuribenzoate reacts with sulfhydryl group of cystein
residues in ATCase resulting in separation of subunits.
-Mercurials dissociate ATCase into two kinds of subunit. How?
-The subunits separated by ion-exchange chromatography or by
ultracentrifugation in a sucrose density gradient.
-The larger subunit : catalytic subunit(c). No CTP binding and no sigmoidal
kinetics
-The smaller subunit : regulatory subunit(r). No catalytic activity
2 c3(trimer) + 3 r2(dimer) → c6r6
Allosteric interactions in ATCase are mediated by large
changes in quaternary structure
1RAI.pdb
• Two catalytic trimers are stacked one on top of the other,
linked by three dimers of the regulatory chains.
• How to mercurial compound dissociate ATCase into two kinds of
subunit? – zinc bound to four cysteines, destabilize r-subunits
2IPO.pdb
PALA : bisubstrate (two substrates) analog, competitive inhibitor of
ATCase. binds to the active sites and blocks ATCase.
※ ACTase was crystallized in the presence of PALA.
PALA
PALA binds at sites lying at the bounderies between pairs of c
chains within a catalytic trimer.
- Conformational change : catalytic trimers move 12Å farther apart,
rotate 10 degrees about their threefold axis of symmetry and dimers
rotate 15 degrees when PALA(substrate analog) bound.
In the absence of substrate
T(tense) state
In the presence of substrate
R(relaxed) state
The enzyme exists in an equilibrium
between the T and R state.
• In the absence of substrate, almost all the
enzyme molecules are in the T state(low affinity
for substrates and low catalytic activity).
• Occasional substrate binding to active site →
entire enzyme shifts to the R state(higher
binding affinity).
• Mechanism for allosteric regulation
1). Concerted mechanism
- the change in the enzyme is ‘all or none’
(explains the behavior of ATCase well)
2). Sequential model
- the binding of ligand to one site on the complex can affect
neighboring sites without T-toR- transition
※ Most other allosteric enzymes have features of both
models.
High Km
low Km
- Mixture of two Michaelis-Menten enzymes.
-As the concentration of substrate is increased, the equilibrium shifts from the T
state to the R state.
-Homotrophic effect by substrate; benefical when small change in substance conc
is physiologically important
Allosteric regulators modulate the T-to-R
equilibrium
- The enzyme is in the T state when bound
to CTP.
-Binding sites for CTP exist in regulatory
chains which are far away from each active
sites.
-How can CTP inhibit the catalytic activity
of the enzyme?
In the absence of any substrate
-CTP stabilize the T state.
-The binding of CTP shifts the equilibrium toward the T state and
makes it more difficult for substrate binding to convert the enzyme
into the R state.
In presence of CTP, more substrate
is required to attain a given reaction
rate.
In presence of ATP, the reaction
rate is increased.
High levels of ATP prevent CTP from inhibiting the
enzyme. Nonsubstrate effects on allosteric enzyme
Heterotropic effects
• Physiological roles of the increase in ATCase
activity in response to increased ATP
concentration
1) High ATP concentration signals a high concentration of
purine nucleotides in the cell. (to balance between purine
and pyrimidine)
2) A high concentration of ATP indicates that energy is
available for mRNA synthesis and DNA replication and lead
to the synthesis of pyrimidines needed for these
processes.
Equilibrium between the T and the R state
R↔T
L = [T]/[R]
L = the equilibrium constant between the R and the T
forms
CTP-saturated form, the
value of L increases from 250
to 1250.
For the ATP saturated form,
the value of L decreases to
70.
10.2 Isozymes provide a means of regulation specific
to distinct tissues and developmental states
• Isozymes (or isoenzymes) are enzymes that differ in amino
acid sequence yet catalyze the same reaction.
- have Different Km, different regulatory molecules
-LDH(lactate dehydrogenase): catalyzes a step in anaerobic glucose
metabolism and glucose synthesis (pyruvate  lactate ch16 참조).
-H isozyme : expressed in heart muscle.
-M isozyme : expressed in skeletal muscle.
amino acid sequences are 75% identical each other.
H isozyme (square) has higher affinity for substrate than M (circle)
The rat heart LDH isozyme profile changes in the course of
development. Functional enzyme is tetrameric.
1. Sequential displacement
- Ordered.
Lactate dehydrogenase
①
②
①
②
- The coenzyme always binds first and the lactase is
always released first.
LDH isozyme content varies by tissue.
-The M4 isozyme functions optimally in the anaerobic environment of
skeletal muscle, whereas the H4 isozyme does in the aerobic
environment of heart muscle.
Myocardial infarction, heart attack; H4 to H3M ratio increase in blood
10.3 Covalent modification is a means of regulating
enzyme activity
Some modifications are reversible.
Phosphorylation is a highly effective means of regulating
the activities of target proteins
- 30% of eukaryotic proteins are phosphorylated.
-The enzymes catalyzing phosphorylation reactions are called
protein kinases.
-ATP is the most common donor of phosphoryl group.
γ
β
α
-Protein phosphatases reverse the effects of kinases by catalyzing
the removal of phosphoryl groups attacked to proteins.
-Hydroxyl-containing side chain is regenerated and Pi is produced.
-Vital role in cells because the enzymes turn off the signals
-One class of highly conserved phosphatase called PP2A
suppresses the cancer-promoting activity of certain kinases.
Kinase
Phosphatase
- The phosphorylation and dephosphorylation are not the reverse of
one another; irreversible under physiological conditions without
enzymes
-With only the help of kinases and phosphatase, take place
-The rate of cycling between the phosphorylated and the
dephosphorylated states depends on the relative activities of kinases
and phosphatases
Phosphorylation is a highly effective means of controlling
the activity of proteins for several reasons;
1) A phosphoryl group adds two negative charges to a modified protein. New
electrostatic interactions
2) A phosphoryl group can form three or more hydrogen bonds.
3) The free energy of phosphorylation is large. (-12kcalmol-1 by ATP, half is
consumed in making phosphorylation; half (6kcalmol-1) is conserved in the
phosphorylated protein. Phosphorylation can change the conformational
equilibrium by the order of 104 ; 1.4kcalmol-1 correspond to 10 fold
increase in an equilibrium constant)
4) Phosphorylation and dephosphorylation can take place in less than a
second or over a span of hours.
5) Phosphorylation often highly amplify signaling effects.
6) ATP is the cellular energy currency. Links the energy status of the cell to
the regulation of metabolism.
Cyclic AMP activates protein kinase A (PKA) by
altering the quaternary structure
-Flight or fight; epinephrine (adrenaline)
triggers cAMP formation
-Cyclic AMP subsequently activates a
key enzyme: protein kinase A.
-The kinase alters the activities of target
proteins by phosphorylating specific
serine or threonine residues.
The consensus sequence recognized by PKA is R-R-X-S/T-Z
; (X, small residues; Z, large hydrophobic one)
-PKA provides a clear example of the integration of allosteric
regulation and phosphorylation.
-PKA consists of two kinds of subunit: a 49kDa regulatory(R) and a
38kDa catalytic(C) subunit.
In the absence of cAMP, R and C
form complex. Pseudosubstrate
sequence inhibit catalytic subunit.
In the presence of cAMP, R and C
dissociate from complex.
Catalytic subunit is activated.
ATP and the target protein bind to a deep cleft in the
catalytic subunit of protein kinase A
-The 350-residue catalytic
subunit has two lobes.
-ATP and part of the inhibitor fill
a deep cleft between lobes.
-The two lobes move closer to
one another on substrate binding.
The pseudosubstrate seq. : Arg-Arg-Asn-Ala-Ile
- Arg(pseudosubstrate) and Glu(PKA) form an ion pair.
- Ile(pseudosubstrate) hydrophobically interact with leu(KPA)
10.4 Many enzymes are activated by specific
proteolytic cleavage
Specific proteolysis is a common means of activating enzymes and
other proteins in biological systems. Ex) ;
1) Digestive enzymes
2) Blood clotting
3) Hormones; proinsuline
4) Collagen; procollagen
5) Developmental processes: procollagenase (tadpole, 출산)
6). Programmed cell death, apoptosis: procaspases
The inactive precursor is called a zymogen or a
proenzyme.Proteolytic activation occurs just once.
Chymotypsinogen is activated by specific cleavage of a
single peptide bond
-Chymotrypsin : digestive enzyme in the small
intestine.
-Synthsized as inactive precursor, chymotrypsinogen.
-Enzymes are synthesized in the acinar cells of the
pancreas and stored inside membrane-bounded
granules.
-The cell is stimulated by hormone or nerve, the
granules are released into a duct leading to the
duodenum (십이지장)
Chymotrypsinogen
-Single polypeptide of 245
amino acids is cleaved by
trypsin.
Linked by interchain disulfide bonds
Proteolytic activation of chymotrypsinogen leads
to the formation of a substrate-binding site
-The cleavage of the peptide bond between
amino acid 15 and 16 triggers key
conformational changes.
-Alteration of the position of Ile16.
-The electrostatic interaction between Ile16
and Asp 194 is essential for the structure of
active chymotrypsin.
-Substrate binding cavity and the oxyanion hole are formed after proteolytic
cleavage.
The generation of typsin from trypsinogen leads
to the activation of other zymogens
from the duodenum cells (십이지장)
Cleave lysine-isoleucine peptide
- The formation of trypsin by enteropeptidase is the master
activation step.
Some proteolytic enzymes have specific inhibitors
Pancreatic trypsin inhibitor :
- 6kda
- Inhibits trypsin by binding- very
tightly to its active site. Turnover
rate is very low
- Lys 15 interact with specificity
pocket of trypsin (Asp189).
Trypsin inhibition → prevention of tissue damage of pancreas or
pancreatic ducts.
※ α1-Antitrypsin: protease inhibitor of elastase, a secretary product of
neurophils.
- Block the elastases by binding to the active sites nearly irreversibly.
Antielastase?
- Genetic disorder of α1-Antitrypsin : emphysema (low α1-Antitrypsin
concentraion in the serum – only 15% → cannot inhibit elastase →
elastase destroys alveilar walls in the lung)
Oxidation of methionine 358 of
inhibitor (by smoking)
→ cannot bind to elastase
→ lung damage
Blood clotting is accomplished by a cascade of
zymogen activations
Exposure of anionic surfaces
-Blood clots are formed by a
cascade of zymogen activations.
Amplification!
rapid response to trauma
-After the tissue factor is exposed,
small amounts of thrombin, the
key protease in clotting, are
generated.
-Thrombin activates enzymes and
factors that lead to the generation
of yet more thrombin (= positive
feedback)
-혈우병?
Integral membrane
glycoprotein
Signal
amplified
Transglutaminase
Fibrinogen is converted by thrombin into a fibrin clot
Rod : triple-stranded α–helical coiled coil
6 chain
= 340kda
- Fibrinogen: is made up of three globular units connected by two
rods.
-Thrombin cleaves four arg-gly peptide bond in the central region of
fibrinogen.
-The cleaved peptide of Aα and Bβ chain = fibrinopeptide
-result in (αβγ)2
-Fibirin monomers assemble into ordered fibrous arrays called
fibrin.
-Fibrin has a periodic structure that repeats every 23nm.
- β Domain is specific for sequences of the form gly-his-arg.
- γ Domain binds gly-pro-arg.
-The knob of the α subunits fit into the holes on the γ subunits of another
monomer to form a protofibiril, which is extended when the knobs of the b
subunits fit into the holes of b subunits of other protofibrils.
Prothrombin is readied for activation by a vitamin
K-dependent modification
-Thrombin is synthesized as a zymogen called prothrombin.
-Inactive form: 4 major domains.
①
②
-Activation is begun by proteolytic cleavage of the bond between
arg274 and thr275
-Cleavage of the bond between arg323 and ile324 yield active
thrombin.
※ Vitamin K (deficiency in this vitamin results in defective
blood koagluation)
-Essential for the synthesis of prothrombin and other clotting
factor.
※ Dicoumarol : vitamin K mimic antagonist.
- Binding of discoumarol results in synthesize an abnormal
prothrombin that does not bind Ca2+(cofactor). Strange? Why?
-Amino acid analysis  same A.A sequence Why?
-NMR!
-Normal prothrombin is carboxylated to γ–carboxyglutamate by a vitamin Kdependent enzyme.
- γ–carboxyglutamate
can interact with Ca2+,which anchor
prothrombin phospholipid membrane.
-brings the zymogen into close
proximity to 2 clotting proteins converting into thrombin
-During activation, calcium-binding
domain is removed, freeing the thrombin
from the membrane so that it can
cleave fibrinogen and other targets.
γ–carboxyglutamate
Hemophilia revealed an early step in clotting
-Hemophilia : the best known clotting defect.
sex-linked recessive characteristic.
-In classic hemophilia, factor VIII of the intrinsic pathway is
missing or has markedly reduced activity.
factor VIII is not a protease but it
stimulate the activation of factor X by
factor Ixa
-Recombinant factor VIII
-The activity of factor VIII is markedly
increased by limited proteolysis by
thrombin  positive feed back
The clotting process must be precisely regulated
-Clots must form rapidly.
-Activated clotting factors are short-lived because they diluted by
blood flow, removed by the liver, and degraded by proteases.
-Factor V and VIII are digested by protein C, switched on by the
action of thrombin
-Thrombin has a dual function: catalyzes the formation of fibrin and
it initiates the deactivation of the clotting cascade
Specific inhibitors of clotting factors are also critical.
-Tissue factor pathway inhibitor (TFPI) inhibit the complex of TF-VII-X
-Antithrombin III: a plasma protein, inactivates thrombin by forming an
irreversible complex with it. Blocks clotting factors such as (9,10,11,12).
-Antithrombin III activity is enhanced by heparin (a negatively charged
polysaccharide found in master cells and endothelial cells)
- Heparin is anticoagulant by increase the rate of formation of irreversible
complexes between antithrombin III and clotting factors
-M358R mutation in a1-antitrypsin’s binding pocket change specificity from an
elastase inhibitor to a thrombin inhibitor. a1-antitrypsin increases markedly after
injury to counteract excess elastase. The mutant a1-antitrypsin caused the patient’s
thrombin activity to drop to the level of hemorrhage.
Clots are not permanent structures. How to desolve clots?
-Fibrin is split by plasmin, a serine protease that hydrolyzes peptide
bonds in the coiled coil.
-Plasmin is formed by the proteolytic activation of plasminogen, that
has high affinity for clot.
-This conversion is carried out by tissue-type plasminogen activator
(TPA), of which domain structure is closely related to prothrombin
-TPA bound to fibrin clots swiftly activate adhering plasminogen but
very slowly free plasminogen
Plasminogen -> plasmin -> fibrin degradation -> clot remove
By TPA administered intravenously
Before
After 3 hour treatment TPA
TPA leads to the dissolution of blood clots, as shown by X-ray
images of blood vessels in the heart coronary artery.
So fast!