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 ATCase-

initiated 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 Michaelis-Menten 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

• 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

1RAI.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.

2IPO.pdb

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 substrateT(tense) state

In the presence of substrateR(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.

- 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 substrate

conc is physiologically important

High Km

low Km

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?

-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 the absence of any substrate

In presence of CTP, more substrate is required to attain a given reaction rate.

In presence of ATP, the reaction rate is increased.

Heterotropic effects

High levels of ATP prevent CTP from inhibiting the enzyme. Nonsubstrate effects on allosteric enzyme

• 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.

• Isozymes (or isoenzymes) are enzymes that differ in amino acid sequence yet catalyze the same reaction.

- have Different Km, different regulatory molecules

10.2 Isozymes provide a means of regulation specific to distinct tissues and developmental states

The rat heart LDH isozyme profile changes in the course of development. Functional enzyme is tetrameric.

-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)

1. Sequential displacement - Ordered.

- The coenzyme always binds first and the lactase is always released first.

① ①② ②

Lactate dehydrogenase

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. αβγ

-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.

- 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

Kinase

Phosphatase

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 enzymes2) Blood clotting3) Hormones; proinsuline4) Collagen; procollagen5) 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

- The formation of trypsin by

enteropeptidase is the master activation

step.

from the duodenum cells ( 십이지장 )Cleave lysine-isoleucine peptide

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

-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 ?

Signal amplified

Exposure of anionic surfaces

Integral membrane glycoprotein

Transglutaminase

Fibrinogen is converted by thrombin into a fibrin clot

- 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

6 chain= 340kda

Rod : triple-stranded α–helical coiled coil

-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 subunits fit

into the holes of 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. -γ–carboxyglutamate rich domain (Gla domain)

-Activation is begun by proteolytic cleavage

of the bond between arg274 and thr275

-Cleavage of the bond between arg323 and

ile324 yield active thrombin.

① ②

※ 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. Spoiled clover - 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!-Vitamin K-dependent γ–carboxylation.

-Normal prothrombin is carboxylated to γ–carboxyglutamate by a vitamin K-dependent enzyme.

- γ–carboxyglutamate can interact with Ca2+,which anchorprothrombin phospholipid membrane.-brings the zymogen into closeproximity to 2 clotting proteins converting into thrombin-During activation, calcium-bindingdomain is removed, freeing the thrombinfrom the membrane so that it cancleave 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 in1-antitrypsin’s binding pocket

change specificity from an elastase inhibitor to a

thrombin inhibitor. 1-antitrypsin increases markedly

after injury to counteract excess elastase. The mutant

1-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 removeBy 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!