Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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Figure 10.42. Structures of Vitamin K and Two Antagonists, Dicoumarol and Warfarin.

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin 10.5. Many Enzymes Are Activated by Specific Proteolytic Cleavage

Figure 10.43. The Calcium-Binding Region of Prothrombin. Prothrombin binds calcium ions with the modified

amino acid γ-carboxyglutamate (red).

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin 10.5. Many Enzymes Are Activated by Specific Proteolytic Cleavage

Figure 10.44. Action of Antihemophilic Factor. Antihemophilic factor (VIII) stimulates the activation of factor X by

factor IX

. It is interesting to note that the activity of factor VIII is markedly increased by limited proteolysis by

thrombin and factor X

. This positive feedback amplifies the clotting signal and accelerates clot formation after a

threshold has been reached.

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin 10.5. Many Enzymes Are Activated by Specific Proteolytic Cleavage

Figure 10.45. Electron Micrograph of a Mast Cell. Heparin and other molecules in the dense granules are released

into the extracellular space when the cell is triggered to secrete. [Courtesy of Lynne Mercer.]

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin 10.5. Many Enzymes Are Activated by Specific Proteolytic Cleavage

Figure 10.46. Modular Structure of Tissue-Type Plasminogen Activator (TPA).

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin 10.5. Many Enzymes Are Activated by Specific Proteolytic Cleavage

Figure 10.47. The Effect of Tissue-Type Plasminogen Factor. TPA leads to the dissolution of blood clots, as shown

by x-ray images of blood vessels in the heart (A) before and (B) 3 hours after the administration of TPA. The position of

the clot is marked by the arrow in part A. [After F. Van de Werf, P. A. Ludbrook, S. R. Bergmann, A. J. Tiefenbrunn, K.

A. A. Fox, H. de Geest, M. Verstraete, D. Collen, and B. E. Sobel. New Engl. J. Med. 310(1984):609.]

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin

Summary

Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its

Pathway

Allosteric proteins constitute an important class of proteins whose biological activity can be regulated. Specific

regulatory molecules can modulate the activity of allosteric proteins by binding to distinct regulatory sites, separate from

the functional site. These proteins have multiple functional sites, which display cooperation as evidenced by a sigmoidal

dependence of function on substrate concentration. Aspartate transcarbamoylase (ATCase), one of the best-understood

allosteric enzymes, catalyzes the synthesis of N-carbamoylaspartate, the first intermediate in the synthesis of

pyrimidines. ATCase is feedback inhibited by cytidine triphosphate(CTP), the final product of the pathway. ATP

reverses this inhibition. ATCase consists of separable catalytic (c

) subunits (which bind the substrates) and regulatory

) subunits (which bind CTP and ATP). The inhibitory effect of CTP, the stimulatory action of ATP, and the

cooperative binding of substrates are mediated by large changes in quaternary structure. On binding substrates, the c

subunits of the c

enzyme move apart and reorient themselves. This allosteric transition is highly concerted, as

postulated by the Monod-Wyman-Changeux (MWC) model. All subunits of an ATCase molecule simultaneously

interconvert from the T (low-affinity) to the R (high-affinity) state.

Hemoglobin Transports Oxygen Efficiently by Binding Oxygen Cooperatively

Hemoglobin, the oxygen carrier in the blood, is an allosteric protein. Hemoglobin consists of four polypeptide chains,

each with a heme group

a substituted porphyrin with a central iron. Hemoglobin A, the predominant hemoglobin in

adults, has the subunit structure α

. Hemoglobin transports H

and CO

in addition to O

. Hemoglobin exhibits

three kinds of allosteric effects. First, the oxygen-binding curve of hemoglobin is sigmoidal, which indicates that the

binding of oxygen is cooperative. The binding of oxygen to one heme group facilitates the binding of oxygen to the other

heme groups in the same molecule. Second, the binding of H

and CO

promotes the release of O

from hemoglobin, an

effect that is physiologically important in enhancing the release of O

in metabolically active tissues such as muscle.

These allosteric linkages between the binding of H

, CO

, and O

are known as the Bohr effect. Third, the affinity of

hemoglobin for O

is further regulated by 2,3-bisphosphoglycerate (2,3-BPG), a small molecule with a very high density

of negative charge. 2,3-Bisphosphoglycerate binds tightly to deoxyhemoglobin but not to oxyhemoglobin. Hence, 2,3-

BPG lowers the oxygen affinity of hemoglobin. Fetal hemoglobin (α

) has a higher oxygen affinity than human adult

hemoglobin because fetal hemoglobin binds 2,3-BPG less tightly. Neither the sequential nor the concerted model

completely describes the allosteric behavior of hemoglobin. Rather, the behavior of hemoglobin is best described by a

combined model that employs features of both models.

Isozymes Provide a Means of Regulation Specific to Distinct Tissues and

Developmental Stages

Isozymes differ in structural characteristics but catalyze the same reaction. They provide a means of fine-tuning

metabolism to meet the needs of a given tissue or developmental stage. The results of gene-duplication events provide

the means for subtle regulation of enzyme function.

Covalent Modification Is a Means of Regulating Enzyme Activity

Covalent modification of proteins is a potent means of controlling the activity of enzymes and other proteins.

Phosphorylation is the most common type of reversible covalent modification. Signals can be highly amplified by

phosphorylation because a single kinase can act on many target molecules. The regulatory actions of protein kinases are

reversed by protein phosphatases, which catalyze the hydrolysis of attached phosphoryl groups.

Cyclic AMP serves as an intracellular messenger in the transduction of many hormonal and sensory stimuli. Cyclic AMP

switches on protein kinase A (PKA), a major multifunctional kinase, by binding to the regulatory subunit of the enzyme,

thereby releasing the active catalytic subunits of PKA. In the absence of cAMP, the catalytic sites of PKA are occupied

by pseudosubstrate sequences of the regulatory subunit.

Many Enzymes Are Activated by Specific Proteolytic Cleavage

The activation of an enzyme by proteolytic cleavage of one or a few peptide bonds is a recurring control mechanism seen

in processes as diverse as the activation of digestive enzymes and blood clotting. The inactive precursor is called a

zymogen (or a proenzyme). Trypsinogen is activated by enteropeptidase or trypsin, and trypsin then activates a host of

other zymogens, leading to the digestion of foodstuffs. For instance, trypsin converts chymotrypsinogen, a zymogen, into

active chymotrypsin by hydrolyzing a single peptide bond.

A striking feature of the clotting process is that it is accomplished by a cascade of zymogen conversions, in which the

activated form of one clotting factor catalyzes the activation of the next precursor. Many of the activated clotting factors

are serine proteases. In the final step of clot formation, fibrinogen, a highly soluble molecule in the plasma, is converted

by thrombin into fibrin by the hydrolysis of four arginine-glycine bonds. The resulting fibrin monomer spontaneously

forms long, insoluble fibers called fibrin. Zymogen activation is also essential in the lysis of clots. Plasminogen is

converted into plasmin, a serine protease that cleaves fibrin, by tissue-type plasminogen activator (TPA). Although

zymogen activation is irreversible, specific inhibitors of some proteases exert control. The irreversible protein inhibitor

antithrombin III holds blood clotting in check in the clotting cascade.

Key Terms

feedback (end-product) inhibition

regulatory site

concerted mechanism

cooperativity

homotropic effect

heterotropic effect

sequential model

heme

protoporphyrin

Bohr effect

isozyme (isoenzyme)

covalent modification

protein kinase

protein phosphatase

consensus sequence

protein kinase A (PKA)

pseudosubstrate sequence

zymogen (proenzyme)

enzymatic cascade

intrinsic clotting pathway

extrinsic clotting pathway

I. The Molecular Design of Life 10. Regulatory Strategies: Enzymes and Hemoglobin

Problems

Activity profile. A histidine residue in the active site of aspartate transcarbamoylase is thought to be important in

stabilizing the transition state of the bound substrates. Predict the pH dependence of the catalytic rate, assuming that

this interaction is essential and dominates the pH-activity profile of the enzyme.

See answer

Allosteric switching. A substrate binds 100 times as tightly to the R state of an allosteric enzyme as to its T state.

Assume that the concerted (MWC) model applies to this enzyme.

See answer

(a) By what factor does the binding of one substrate molecule per enzyme molecule alter the ratio of the concentrations

of enzyme molecules in the R and T states?

(b) Suppose that L, the ratio of [T] to [R] in the absence of substrate, is 10

and that the enzyme contains four binding

sites for substrate. What is the ratio of enzyme molecules in the R state to that in the T state in the presence of

saturating amounts of substrate, assuming that the concerted model is obeyed?

Allosteric transition. Consider an allosteric protein that obeys the concerted model. Suppose that the ratio of T to R

formed in the absence of ligand is 10

, K

= 2 mM, and K

= 5 µM. The protein contains four binding sites for

ligand. What is the fraction of molecules in the R form when 0, 1, 2, 3, and 4 ligands are bound?

See answer

Negative cooperativity. You have isolated a dimeric enzyme that contains two identical active sites. The binding of

substrate to one active site decreases the substrate affinity of the other active site. Which allosteric model best

accounts for this negative cooperativity?

See answer

Paradoxical at first glance. Recall that phosphonacetyl-l-aspartate (PALA) is a potent inhibitor of ATCase because

it mimics the two physiological substrates. However, low concentrations of this unreactive bisubstrate analog

increase the reaction velocity. On the addition of PALA, the reaction rate increases until an average of three

molecules of PALA are bound per molecule of enzyme. This maximal velocity is 17-fold as great as it is in the

absence of PALA. The reaction rate then decreases to nearly zero on the addition of three more molecules of PALA

per molecule of enzyme. Why do low concentrations of PALA activate ATCase?

See answer

R versus T. An allosteric enzyme that follows the concerted mechanism has a T/R ratio of 300 in the absence of

substrate. Suppose that a mutation reversed the ratio. How would this mutation affect the relation between the rate of

the reaction and substrate concentration?

See answer

Tuning oxygen affinity. What is the effect of each of the following treatments on the oxygen affinity of hemoglobin

A in vitro?

(a) Increase in pH from 7.2 to 7.4.

(b) Increase in pCO

from 10 to 40 torr.

-4

to 8 × 10

-4

(d) Dissociation of α

into monomeric subunits.

See answer

Avian and reptilian counterparts. The erythrocytes of birds and turtles contain a regulatory molecule different from

2,3-BPG. This substance is also effective in reducing the oxygen affinity of human hemoglobin stripped of 2,3-BPG.

Which of the following substances would you predict to be most effective in this regard?

(a) Glucose 6-phosphate

(b) Inositol hexaphosphate

(d) Malonate

(e) Arginine

(f) Lactate

See answer

Tuning proton affinity. The pK of an acid depends partly on its environment. Predict the effect of the following

environmental changes on the pK of a glutamic acid side chain.

(a) A lysine side chain is brought into close proximity.

(b) The terminal carboxyl group of the protein is brought into close proximity.

See answer

10.

Zymogen activation. When very low concentrations of pepsinogen are added to acidic media, how does the half-

time for activation depend on zymogen concentration?

See answer

11.

A revealing assay. Suppose that you have just examined a young boy with a bleeding disorder highly suggestive of

classic hemophilia (factor VIII deficiency). Because of the late hour, the laboratory that carries out specialized

coagulation assays is closed. However, you happen to have a sample of blood from a classic hemophiliac whom

you admitted to the hospital an hour earlier. What is the simplest and most rapid test that you can perform to

determine whether your present patient also is deficient in factor VIII activity?

See answer

12.

Counterpoint. The synthesis of factor X, like that of prothrombin, requires vitamin K. Factor X also contains γ-

carboxyglutamate residues in its amino-terminal region. However, activated factor X, in contrast with thrombin,

retains this region of the molecule. What is a likely functional consequence of this difference between the two

activated species?

See answer

13.

A discerning inhibitor. Antithrombin III forms an irreversible complex with thrombin but not with prothrombin.

What is the most likely reason for this difference in reactivity?

See answer

14.

Repeating heptads. Each of the three types of fibrin chains contains repeating heptapeptide units (abcdefg) in which

residues a and d are hydrophobic. Propose a reason for this regularity.

See answer

15.

Drug design. A drug company has decided to use recombinant DNA methods to prepare a modified α

-antitrypsin

that will be more resistant to oxidation than is the naturally occurring inhibitor. Which single amino acid

substitution would you recommend?

See answer

Data Interpretation Problems

16.

Distinguishing between models. The graph below shows the fraction of an allosteric enzyme in the R state (f

) and

the fraction of active sites bound to substrate (Y) as a function of substrate concentration. Which model, the

concerted or sequential, best explains these results?

[From M.W. Kirschner and H.K. Schachman, Biochemistry 12 (1966):2997.]

See answer

17.

Reporting live from ATCase. ATCase was reacted with nitromethane to form a colored nitrotyrosine group (λ

max

430 nm) in each of its catalytic chains. The absorption by this reporter group depends on its immediate

environment. An essential lysine residue at each catalytic site also was modified to block the binding of substrate.

Catalytic trimers from this doubly modified enzyme were then combined with native trimers to form a hybrid

enzyme. The absorption by the nitrotyrosine group was measured on addition of the substrate analog succinate.

What is the significance of the alteration in the absorbance at 430 nm?

[After H.K. Schachman. J. Biol. Chem. 263 (1988):18583]

The binding of succinate, an unreactive analog of aspartate, to one trimer changed the visible absorption spectrum

of nitrotyrosine residues attached to the other trimer.

See answer

18.

Reporting live from ATCase 2. A different ATCase hybrid was constructed to test the effects of allosteric activators

and inhibitors. Normal regulatory subunits were combined with nitrotyrosine-containing catalytic subunits. The

addition of ATP in the absence of substrate increased the absorbance at 430 nm, the same change elicited by the

addition of succinate (see the graph in Problem 17). Conversely, CTP in the absence of substrate decreased the

absorbance at 430 nm. What is the significance of the changes in absorption of the reporter groups?.

[After H.K. Schachman. J. Biol. Chem. 263(1988):18583.]

See answer

Chapter Integration Problem

19.

Sticky patches. The substitution of valine for glutamate at position 6 of the β chains of hemoglobin places a

nonpolar residue on the outside of hemoglobin S, the version of hemoglobin responsible for sickle-cell anemia. The

oxygen affinity and allosteric properties of hemoglobin are virtually unaffected by this change. However, the valine

side chain of hemoglobin S interacts with a complementary sticky patch (formed by phenyl-alanine β85 and leucine

β88) on another hemoglobin molecule

a patch that is exposed in deoxygenated but not in oxygenated

hemoglobin. What is the chemical basis for the interaction between the hemoglobin molecules? What would be the

effect of this interaction?

See answer

Mechanism Problems

20.

Aspartate transcarbamoylase. Write the mechanism (in detail) for the conversion of aspartate and carbamoyl

phosphate into N-carbamoylaspartate. Include a role for the residue histidine present in the active site.

See answer

21.

Protein kinases. Write a mechanism (in detail) for the phosphorylation of a serine residue by ATP catalyzed by a

protein kinase. What groups might you expect to find in the enzyme active site?

See answer

Media Problem