Harper Regulacija Enzima

8/10/2019 Harper Regulacija Enzima

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CHAPTER

9

Enzymes: Regulation of Activities

Peter J. Kennelly, PhD & Victor W. Rodwell, PhD

OBJECTIVES

After studying this chapter, you should be able to:

Explain the concept of whole-body homeostasis and its response to fluctuations in

the external environment.

Discuss why the cellular concentrations of substrates for most enzymes tend to be

close toKm.

List multiple mechanisms by which active control of metabolite flux is achieved.

Describe the advantages of certain enzymes being elaborated as proenzymes.

Illustrate the physiologic events that trigger the conversion of a proenzyme to the

corresponding active enzyme.

Describe typical structural changes that accompany conversion of a proenzyme to

the active enzyme.

Describe the basic features of a typical binding site for metabolites and second

messengers that regulate catalytic activity of certain enzymes.

Indicate two general ways in which an allosteric effector can modify catalytic

activity.Outline the roles of protein kinases, protein phosphatases, and of regulatory and

hormonal and second messengers in initiating a metabolic process.

BIOMEDICAL IMPORTANCE

The nineteenth-century physiologist Claude Bernard enunciated the conceptual basis

for metabolic regulation. He observed that living organisms respond in ways that are

both quantitatively and temporally appropriate to permit them to survive the multiple

challenges posed by changes in their external and internal environments. Walter

Cannon subsequently coined the term homeostasis to describe the ability of animals

to maintain a constant intracellular environment despite changes in their externalenvironment. We now know that organisms respond to changes in their external and

internal environment by balanced, coordinated adjustments in the rates of specific

metabolic reactions. Perturbations of the sensor-response machinery responsible for

maintaining homeostatic balance can be deleterious to human health. Cancer,

diabetes, cystic fibrosis, and Alzheimers disease, for example, are all characterized

by regulatory dysfunctions triggered by pathogenic agents or genetic mutations. Many


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oncogenic viruses elaborate protein-tyrosine kinases that modify the regulatory events

that control patterns of gene expression, contributing to the initiation and progression

of cancer. The toxin from Vibrio cholerae,the causative agent of cholera, disables

sensor-response pathways in intestinal epithelial cells by ADP-ribosylating the GTP-

binding proteins (G-proteins) that link cell surface receptors to adenylyl cyclase. The

consequent activation of the cyclase leads to the unrestricted flow of water into theintestines, resulting in massive diarrhea and dehydration. Yersinia pestis,the causative

agent of plague, elaborates a protein-tyrosine phosphatase that hydrolyzes phosphoryl

groups on key cytoskeletal proteins. Dysfunctions in the proteolytic systems

responsible for the degradation of defective or abnormal proteins are believed to play

a role in neurodegenerative diseases such as Alzheimer and Parkinsons. In addition to

their immediate function as regulators of enzyme activity, protein degradation, etc,

covalent modifications such as phosphorylation, acetylation, and ubiquitination

provide a protein-based code for the storage and hereditary transmission of

information (Chapter 35). Such DNA-independent information systems are referred to

as epigenetic.Knowledge of factors that control the rates of enzyme-catalyzed

reactions thus is essential to an understanding of the molecular basis of disease and its

transmission. This chapter outlines the patterns by which metabolic processes are

controlled, and provides illustrative examples. Subsequent chapters provide additional

examples.

REGULATION OF METABOLITE FLOW CAN BE ACTIVE OR PASSIVE

Enzymes that operate at their maximal rate cannot respond to increases in substrate

concentration, and can respond only to precipitous decreases in substrate

concentration. TheKmvalues for most enzymes, therefore, tend to be close to theaverage intracellular concentration of their substrates, so that changes in substrate

concentration generate corresponding changes in the metabolite flux (Figure 9

1).Responses to changes in substrate level represent an important butpassivemeans

for coordinating metabolite flow and maintaining homeostasis in quiescent cells.

However, they offer a limited scope for responding to changes in environmental

variables. The mechanisms that regulate enzyme efficiency in an activemanner in

response to internal and external signals are discussed below.
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FIGURE 91 Differential response of the rate of an enzyme-catalyzed reaction,

V, to the same incremental change in substrate concentration at a substrate

concentration close to Km(VA) or far above Km(VB).

Metabolite Flow Tends to Be Unidirectional

Despite the existence of short-term oscillations in metabolite concentrations and

enzyme levels, living cells exist in a dynamic steady state in which the mean

concentrations of metabolic intermediates remain relatively constant over time. While

all chemical reactions are to some extent reversible, in living cells the reaction

products serve as substrates forand are removed byother enzyme-catalyzed

reactions (Figure 92).Many nominally reversible reactions thus occur

unidirectionally. This succession of coupled metabolic reactions is accompanied by an

overall change in free energy that favors unidirectional metabolite flow (Chapter 11).

The unidirectional flow of metabolites through a pathway with a large overallnegative change in free energy is analogous to the flow of water through a pipe in

which one end is lower than the other. Bends or kinks in the pipe simulate individual

enzyme-catalyzed steps with a small negative or positive change in free energy. Flow

of water through the pipe nevertheless remains unidirectional due to the overall

change in height, which corresponds to the overall change in free energy in a

pathway (Figure 93).
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disfavored steps from glycolysis are replaced by new reactions catalyzed by distinct

enzymes (Chapter 20).

The ability of enzymes to discriminate between the structurally similar coenzymes

NAD+and NADP+also results in a form of compartmentation. The reduced forms of

both coenzymes are not readily distinguishable. However, the reactions that generate

and later consume electrons that are destined for ATP generation are segregated in

NADH, away from those used in the reductive steps of many biosynthetic pathways,

which are carried by NADPH.

Controlling an Enzyme That Catalyzes a Rate-Limiting Reaction Regulates an Entire Metabolic Pathway

While the flux of metabolites through metabolic pathways involves catalysis by

numerous enzymes, active control of homeostasis is achieved by the regulation of

only a select subset of these enzymes. The ideal enzyme for regulatory intervention is

one whose quantity or catalytic efficiency dictates that the reaction it catalyzes is slow

relative to all others in the pathway. Decreasing the catalytic efficiency or the quantity

of the catalyst responsible for the bottleneck orrate-limiting reactionimmediately

reduces metabolite flux through the entire pathway. Conversely, an increase in either

its quantity or catalytic efficiency enhances flux through the pathway as a whole. For

example, acetyl-CoA carboxylase catalyzes the synthesis of malonyl-CoA, the first

committed reaction of fatty acid biosynthesis (Chapter 23). When synthesis of

malonyl-CoA is inhibited, subsequent reactions of fatty acid synthesis cease for lack

of substrates. As natural governors of metabolic flux, the enzymes that catalyze

rate-limiting steps also constitute efficient targets for regulatory intervention by drugs.

For example, statin drugs curtail synthesis of cholesterol by inhibiting HMG-CoA

reductase, which catalyzes the rate-limiting reaction of cholesterogenesis.

REGULATION OF ENZYME QUANTITY

The catalytic capacity of the rate-limiting reaction in a metabolic pathway is the

product of the concentration of enzyme molecules and their intrinsic catalytic

efficiency. It therefore follows that catalytic capacity can be influenced both by

changing the quantity of enzyme present and by altering its intrinsic catalytic

efficiency.

Proteins Are Continuously Synthesized and Degraded

By measuring the rates of incorporation of15

N-labeled amino acids into protein andthe rates of loss of 15N from protein, Schoenheimer deduced that body proteins are in

a state of dynamic equilibrium in which they are continuously synthesized and

degradeda process referred to as protein turnover.This holds even for those

proteins that are present at an essentially constant, or constitutive, steady-state level

over time. On the other hand, the concentrations of many enzymes are influenced by a

wide range of physiologic, hormonal, or dietary factors.
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The absolute quantity of an enzyme reflects the net balance between its rate of

synthesis and its rate of degradation. In human subjects, alterations in the levels of

specific enzymes can be effected by a change in the rate constant for the overall

processes of synthesis (ks), degradation (kdeg), or both.

Control of Enzyme Synthesis

The synthesis of certain enzymes depends upon the presence of inducers, typically

substrates or structurally related compounds that stimulate the transcription of the

gene that encodes them (Chapters 36and37).Escherichia coligrown on glucose will,for example, only catabolize lactose after addition of a -galactoside, an inducer that

triggers synthesis of a -galactosidase and a galactoside permease (Figure 383).

Inducible enzymes of humans include tryptophan pyrrolase, threonine dehydratase,

tyrosine--ketoglutarate aminotransferase, enzymes of the urea cycle, HMG-CoA

reductase, and cytochrome P450. Conversely, an excess of a metabolite may curtail

synthesis of its cognate enzyme via repression.Both induction and repression

involve ciselements, specific DNA sequences located upstream of regulated genes,

and trans-actingregulatory proteins. The molecular mechanisms of induction and

repression are discussed inChapter 38.The synthesis of other enzymes can be

stimulated by the interaction of hormones and other extracellular signals with specificcell-surface receptors. Detailed information on the control of protein synthesis in

response to hormonal stimuli can be found inChapter 42.

Control of Enzyme Degradation

In animals many proteins are degraded by the ubiquitin-proteasome pathway, the

discovery of which earned Aaron Ciechanover, Avram Hershko, and Irwin Rose a

Nobel Prize. Degradation takes place in the 26S proteasome, a large macromolecular

complex made up of more than 30 polypeptide subunits arranged in the form of a

hollow cylinder. The active sites of its proteolytic subunits face the interior of the

cylinder, thus preventing indiscriminate degradation of cellular proteins. Proteins aretargeted to the interior of the proteasome by ubiquitination, the covalent attachment

of one or more ubiquitin molecules. Ubiquitin is a small, approximately 75 residue,

protein that is highly conserved among eukaryotes. Ubiquitination is catalyzed by a

large family of enzymes called E3 ligases, which attach ubiquitin to the side-chain

amino group of lysyl residues.
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The ubiquitin-proteasome pathway is responsible both for the regulated degradation

of selected cellular proteins (for example, cyclinsChapter 35) and for the removal

of defective or aberrant protein species. The key to the versatility and selectivity of

the ubiquitin-proteasome system resides in both the variety of intracellular E3 ligases

and their ability to discriminate between the different physical or conformational

states of target proteins. Thus, the ubiquitin-proteasome pathway can selectivelydegrade proteins whose physical integrity and functional competency have been

compromised by the loss of or damage to a prosthetic group, oxidation of cysteine or

histidine residues, or deamidation of asparagine or glutamine residues. Recognition by

proteolytic enzymes also can be regulated by covalent modifications such as

phosphorylation; binding of substrates or allosteric effectors; or association with

membranes, oligonucleotides, or other proteins. A growing body of evidence suggests

that dysfunctions of the ubiquitin-proteasome pathway contribute to the accumulation

of aberrantly folded protein species characteristic of several neurodegenerative

diseases.

MULTIPLE OPTIONS ARE AVAILABLE FOR REGULATING CATALYTIC

ACTIVITY

In humans the induction of protein synthesis is a complex multistep process that

typically requires hours to produce significant changes in overall enzyme level. By

contrast, changes in intrinsic catalytic efficiency effected by binding of dissociable

ligands (allosteric regulation)or by covalent modificationachieve regulation of

enzymic activity within seconds. Consequently, changes in protein level generally

dominate when meeting long-term adaptive requirements, whereas changes in

catalytic efficiency are best suited for rapid and transient alterations in metaboliteflux.

ALLOSTERIC EFFECTORS REGULATE CERTAIN ENZYMES

Feedback inhibition refers to the process by which the end product of a multistep

biosynthetic pathway binds to and inhibits an enzyme catalyzing one of the early steps

in that pathway. In the following example, for the biosynthesis of D from A catalyzed

by enzymes Enz1through Enz3:

high concentrations of D inhibit the conversion of A to B. In this example, the

feedback inhibitor D acts as a negative allosteric effectorof Enz1. Inhibition results,

not from the backing up of intermediates, but from the ability of D to bind to and

inhibit Enz1. Generally, D binds at an allosteric site, one spatially distinct from the

catalytic site of the target enzyme. Feedback inhibitors thus typically bear little or no
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structural similarity to the substrates of the enzymes they inhibit. For example,

NAD+and 3-phosphogylcerate, the substrates for 3-phosphgylcerate dehydrogenase,

which catalyzes the first committed step in serine biosynthesis, bear no resemblance

to the feedback inhibitor serine. In branched biosynthetic pathways, such as those

responsible for nucleotide biosynthesis (Chapter 33), the initial reactions supply

intermediates required for the synthesis of multiple end products.Figure 94shows ahypothetical branched biosynthetic pathway in which curved arrows lead from

feedback inhibitors to the enzymes whose activity they inhibit. The sequences S3

A, S4 B, S4 C, and S3 D each represent linear reaction sequences that are

feedback-inhibited by their end products. Branch point enzymes thus can be targeted

to route metabolite flow.

FIGURE 94 Sites of feedback inhibition in a branched biosyntheticpathway.S1S5are intermediates in the biosynthesis of end products AD. Straight

arrows represent enzymes catalyzing the indicated conversions. Curved red arrows

represent feedback loops and indicate sites of feedback inhibition by specific end

products.

Feedback inhibitors typically inhibit the first committed step in a particular

biosynthetic sequence. The kinetics of feedback inhibition may be competitive,

noncompetitive, partially competitive, or mixed. Layering multiple feedback loops

can provide additional fine control. For example, as shown inFigure 95,the

presence of excess product B decreases the requirement for substrate S2. However,

S2is also required for synthesis of A, C, and D. Therefore, for this pathway, excess B

curtails synthesis of all four end products, regardless of the need for the other three.

To circumvent this potential difficulty, each end product may only partially inhibit

catalytic activity. The effect of an excess of two or more end products may be strictly

additive or, alternatively, greater than their individual effect (cooperative feedbackinhibition).
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FIGURE 95 Multiple feedback inhibition in a branched biosyntheticpathway.Superimposed on simple feedback loops (dashed red arrows) are multiple

feedback loops (solid red arrows) that regulate enzymes common to biosynthesis of

several end products.

Aspartate Transcarbamoylase Is a Model Allosteric Enzyme

Aspartate transcarbamoylase (ATCase), the catalyst for the first reaction unique to

pyrimidine biosynthesis (Figure 339), is a target of feedback regulation by two

nucleotide triphosphates: cytidine triphosphate (CTP) and adenosine triphosphate.

CTP, an end product of the pyrimidine biosynthetic pathway, inhibits ATCase,

whereas the purine nucleotide ATP activates it. Moreover, high levels of ATP can

overcome inhibition by CTP, enabling synthesis ofpyrimidinenucleotides to proceed

whenpurinenucleotide levels are elevated.

Allosteric & Catalytic Sites Are Spatially Distinct

Jacques Monod proposed the existence of allosteric sites that are physically distinctfrom the catalytic site. He reasoned that the lack of structural similarity between a

feedback inhibitor and the substrate(s) for the enzyme whose activity it regulates

indicated that these effectors are not isostericwith a substrate but allosteric(occupy

another space).Allosteric enzymesthus are those for which catalysis at the active

site may be modulated by the presence of effectors at an allosteric site. The existence

of spatially distinct active and allosteric sites has since been verified in several

enzymes using many lines of evidence. For example, x-ray crystallography revealed

that the ATCase ofE coliconsists of six catalytic subunits and six regulatory subunits,

the latter of which bind the nucleotide triphosphates that modulate activity. In general,

binding of an allosteric regulator induces a conformational change in the enzyme that

encompasses the active site.

Allosteric Effects May Be on Kmor on Vmax

To refer to the kinetics of allosteric inhibition as competitive or noncompetitive

with substrate carries misleading mechanistic implications. We refer instead to two

classes of allosterically regulated enzymes: K-series and V-series enzymes. For K-
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series allosteric enzymes, the substrate saturation kinetics is competitive in the sense

thatKmis raised without an effect on Vmax. For V-series allosteric enzymes, the

allosteric inhibitor lowers Vmaxwithout affecting theKm. Alterations

inKmor Vmaxoften are the product of conformational changes at the catalytic site

induced by binding of the allosteric effector at its site. For a K-series allosteric

enzyme, this conformational change may weaken the bonds between substrate andsubstrate-binding residues. For a V-series allosteric enzyme, the primary effect may

be to alter the orientation or charge of catalytic residues, lowering Vmax. Intermediate

effects onKmand Vmax, however, may be observed consequent to these

conformational changes.

FEEDBACK REGULATION IS NOT SYNONYMOUS WITH FEEDBACK INHIBITION

In both mammalian and bacterial cells, some end products feed back to control their

own synthesis, in many instances by feedback inhibition of an early biosynthetic

enzyme. We must, however, distinguish betweenfeedback regulation, a

phenomenologic term devoid of mechanistic implications, and feedback inhibition, a

mechanism for regulation of enzyme activity. For example, while dietary cholesterol

decreases hepatic synthesis of cholesterol, this feedback regulationdoes not involve

feedback inhibition.HMG-CoA reductase, the rate-limiting enzyme of

cholesterogenesis, is affected, but cholesterol does not inhibit its activity. Rather,

regulation in response to dietary cholesterol involves curtailment by cholesterol or a

cholesterol metabolite of the expression of the gene that encodes HMG-CoA

reductase (enzyme repression) (Chapter 26).

MANY HORMONES ACT THROUGH ALLOSTERIC SECOND MESSENGERS

Nerve impulses and the binding of many hormones to cell surface receptors elicit

changes in the rate of enzyme-catalyzed reactions within target cells by inducing the

release or synthesis of specialized allosteric effectors called second messengers.The

primary, or first, messenger is the hormone molecule or nerve impulse. Second

messengers include 3, 5-cAMP, synthesized from ATP by the enzyme adenylyl

cyclase in response to the hormone epinephrine, and Ca2+, which is stored inside the

endoplasmic reticulum of most cells. Membrane depolarization resulting from a nerve

impulse opens a membrane channel that releases calcium ions into the cytoplasm,

where they bind to and activate enzymes involved in the regulation of muscle

contraction and the mobilization of stored glucose from glycogen. Glucose thensupplies the increased energy demands of muscle contraction. Other second

messengers include 3,5-cGMP, nitric oxide, and the polyphosphoinositols produced

by the hydrolysis of inositol phospholipids by hormone-regulated phospholipases.

Specific examples of the participation of second messengers in the regulation of

cellular processes can be found inChapters 19,42,and48.
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REGULATORY COVALENT MODIFICATIONS CAN BE REVERSIBLE OR

IRREVERSIBLE

In mammalian cells, a wide range of regulatory covalent modifications occur. Partial

proteolysisand phosphorylation, for

example, are frequently employed to regulate the catalytic activity of enzymes. Onthe other hand, histones and other DNA binding proteins in chromatin are subject to

extensive modification by acetylation, methylation, ADP-ribosylation, as well as

phosphorylation. The latter modifications, which modulate the manner in which the

proteins within chromatin interact with each other as well as the DNA itself, constitute

the basis for the histone code. The resulting changes in chromatin structure within

the region affected can render genes more accessible to the protein responsible for

their transcription, thereby enhancing gene expression or, on a larger scale, facilitating

replication of the entire genome (Chapter 38). On the other hand, changes in

chromatin structure that restrict the accessibility of genes to transcription factors,

DNA-dependent RNA polymerases, etc, thereby inhibiting transcription, are saidto silencegene expression.

The histone code represents a classic example of epigenetics, the hereditary

transmission of information by a means other than the sequence of nucleotides that

comprise the genome. In this instance, the pattern of gene expression within a newly

formed daughter cell will be determined, in part, by the particular set of histone

covalent modifications embodied in the chromatin proteins inherited from the

parental cell.

Acetylation, ADP-ribosylation, methylation, and phosphorylation are all examples

of reversible covalent modifications. In this instance, reversible refers to the fact

that the modified protein can be restored to its original, modification-free state. It doesnot, however, refer to the mechanisms by which such restoration takes place.

Thermodynamics dictates that if the enzyme-catalyzed reaction by which the

modification was introduced is thermodynamically favorable, the free energy change

involved in simply trying to run the reaction in reverse will be unfavorable. The

phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by

protein kinases, is thermodynamically favored as a consequence of utilizing the high-

energy gamma phosphoryl group of ATP. Phosphate groups are removed, not by

recombining the phosphate with ADP to form ATP, but by a hydrolytic reaction

catalyzed by enzymes called protein phosphatases. Similarly, acetyltransferases

employ a high-energy donor substrate, NAD+, while deacetylases catalyze a direct

hydrolysis that generates free acetate.

Because the high entropic barrier prevents the reunification of the two portions of a

protein produced by hydrolysis of a peptide bond, proteolysis constitutes a

physiologically irreversible modification. Once a proprotein is activated, it will

continue to carry out its catalytic or other functions until it is removed by degradation
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or some other means. Zymogen activation thus represents a simple and economical,

albeit one way, mechanism for restraining the latent activity of a protein until the

appropriate circumstances are encountered. It is therefore not surprising that partial

proteolysis is employed frequently to regulate proteins that work in the

gastrointestinal tract or bloodstream rather than in the interior of cells.

PROTEASES MAY BE SECRETED AS CATALYTICALLY INACTIVE PROENZYMES

Certain proteins are synthesized and secreted as inactive precursor proteins known

as proproteins.Selective, or partial, proteolysis converts a proprotein by one or

more successive proteolytic clips to a form that exhibits the characteristic activity of

the mature protein, for example, its catalytic activity. The proprotein forms of

enzymes are termed proenzymesor zymogens.Proteins synthesized as proproteins

include the hormone insulin (proprotein = proinsulin), the digestive enzymes pepsin,

trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and

chymotrypsinogen, respectively), several factors of the blood clotting and blood clot

dissolution cascades (seeChapter 51), and the connective tissue protein collagen

(proprotein = procollagen).

Proenzymes Facilitate Rapid Mobilization of an Activity in Response to Physiologic Demand

The synthesis and secretion of proteases as catalytically inactive proenzymes protect

the tissue of origin (eg, the pancreas) from autodigestion, such as can occur in

pancreatitis. Certain physiologic processes such as digestion are intermittent but fairly

regular and predictable in frequency. Others such as blood clot formation, clot

dissolution, and tissue repair are brought on line only in response to pressing

physiologic or pathophysiologic need. The processes of blood clot formation anddissolution clearly must be temporally coordinated to achieve homeostasis. Enzymes

needed intermittently but rapidly often are secreted in an initially inactive form since

new synthesis and secretion of the required proteins might be insufficiently rapid to

respond to a pressing pathophysiologic demand such as the loss of blood (seeChapter

51).

Activation of Prochymotrypsin Requires Selective Proteolysis

Selective proteolysis involves one or more highly specific proteolytic clips that may

or may not be accompanied by separation of the resulting peptides. Most importantly,

selective proteolysis often results in conformational changes that create the catalyticsite of an enzyme. Note that while the catalytically essential residues His 57 and Asp

102 reside on the B peptide of -chymotrypsin, Ser 195 resides on the C

peptide(Figure 96).The conformational changes that accompany selective

proteolysis of prochymotrypsin (chymotrypsinogen) align the three residues of the

charge-relay network (seeFigure 77), forming the catalytic site. Note also that
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contact and catalytic residues can be located on different peptide chains but still be

within bond-forming distance of bound substrate.

FIGURE 96 Two-dimensional representation of the sequence of proteolytic

events that ultimately result in formation of the catalytic site of chymotrypsin,

which includes the Asp 102-His57-Ser195 catalytic triad (seeFigure 7

7).Successive proteolysis forms prochymotrypsin (pro-CT), -chymotrypsin (-Ct),

and ultimately -chymotrypsin (-CT), an active protease whose three peptides (A, B,

C) remain associated by covalent inter-chain disulfide bonds.

REVERSIBLE COVALENT MODIFICATION REGULATES KEY MAMMALIAN

PROTEINS

Mammalian proteins are the targets of a wide range of covalent modification

processes. Modifications such as prenylation, glycosylation, hydroxylation, and fatty

acid acylation introduce unique structural features into newly synthesized proteins that

tend to persist for the lifetime of the protein. Among the covalent modifications that

regulate protein function (eg, methylation, acetylation), the most common by far is

phosphorylationdephosphorylation. Protein kinasesphosphorylate proteins by

catalyzing transfer of the terminal phosphoryl group of ATP to the hydroxyl groups ofseryl, threonyl, or tyrosyl residues, forming O-phosphoseryl, O-phosphothreonyl, or

O-phosphotyrosyl residues, respectively (Figure 97).Some protein kinases target the

side chains of histidyl, lysyl, arginyl, and aspartyl residues. The unmodified form of

the protein can be regenerated by hydrolytic removal of phosphoryl groups, catalyzed

by protein phosphatases.
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FIGURE 97 Covalent modification of a regulated enzyme by phosphorylation

dephosphorylation of a seryl residue.

A typical mammalian cell possesses thousands of phosphorylated proteins and

several hundred protein kinases and protein phosphatases that catalyze their

interconversion. The ease of interconversion of enzymes between their phospho-and

dephospho- forms accounts, in part, for the frequency with which phosphorylation

dephosphorylation is utilized as a mechanism for regulatory control. Phosphorylation

dephosphorylation permits the functional properties of the affected enzyme to be

altered only for as long as it serves a specific need. Once the need has passed, the

enzyme can be converted back to its original form, poised to respond to the next

stimulatory event. A second factor underlying the widespread use of protein

phosphorylationdephosphorylation lies in the chemical properties of the phosphoryl

group itself. In order to alter an enzymes functional properties, any modification of

its chemical structure must influence the proteins three-dimensional configuration.

The high charge density of protein-bound phosphoryl groupsgenerally2 atphysiologic pHand their propensity to form strong salt bridges with arginyl and

lysyl residues renders them potent agents for modifying protein structure and function.

Phosphorylation generally influences an enzymes intrinsic catalytic efficiency or

other properties by inducing conformational changes. Consequently, the amino acids

targeted by phosphorylation can be and typically are relatively distant from the

catalytic site itself.

Covalent Modifications Regulate Metabolite Flow

In many respects, sites of protein phosphorylation and other covalent modifications

can be considered another form of allosteric site. However, in this case, the allostericligand binds covalently to the protein. Both phosphorylation-dephosphorylation and

feedback inhibition provide short-term, readily reversible regulation of metabolite

flow in response to specific physiologic signals. Both act without altering gene

expression. Both act on early enzymes of a protracted biosynthetic metabolic

pathway, and both act at allosteric rather than catalytic sites. Feedback inhibition,

however, involves a single protein and lacks hormonal and neural features. By


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contrast, regulation of mammalian enzymes by phosphorylationdephosphorylation

involves several proteins and ATP, and is under direct neural and hormonal control.

PROTEIN PHOSPHORYLATION IS EXTREMELY VERSATILE

Protein phosphorylationdephosphorylation is a highly versatile and selective process.

Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a

proteins surface, only one or a small subset are targeted. While the most common

enzyme function affected is the proteins catalytic efficiency, phosphorylation can

also alter its location within the cell, susceptibility to proteolytic degradation, or

responsiveness to regulation by allosteric ligands. Phosphorylation can increase an

enzymes catalytic efficiency, converting it to its active form in one protein, while

phosphorylation of another protein converts it to an intrinsically inefficient, or

inactive, form (Table 91).

TABLE 91 Examples of Mammalian Enzymes Whose Catalytic Activity Is

Altered by Covalent Phosphorylation-Dephosphorylation

Many proteins can be phosphorylated at multiple sites. Others are subject to

regulation both by phosphorylationdephosphorylation and by the binding of

allosteric ligands, or by phosphorylationdephosphorylation and another covalent

modification. Phosphorylationdephosphorylation at any one site can be catalyzed by

multiple protein kinases or protein phosphatases. Many protein kinases and most

protein phosphatases act on more than one protein and are themselves interconverted
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between active and inactive forms by the binding of second messengers or by covalent

modification by phosphorylationdephosphorylation.

The interplay between protein kinases and protein phosphatases, between the

functional consequences of phosphorylation at different sites, between

phosphorylation sites and allosteric sites, or between phosphorylation sites and other

sites of covalent modification provides the basis for regulatory networks that integrate

multiple environmental input signals to evoke an appropriate coordinated cellular

response. In these sophisticated regulatory networks, individual enzymes respond to

different environmental signals. For example, if an enzyme can be phosphorylated at a

single site by more than one protein kinase, it can be converted from a catalytically

efficient to an inefficient (inactive) form, or vice versa, in response to any one of

several signals. If the protein kinase is activated in response to a signal different from

the signal that activates the protein phosphatase, the phosphoprotein becomes a

decision node. The functional output, generally catalytic activity, reflects the

phosphorylation state. This state or degree of phosphorylation is determined by therelative activities of the protein kinase and protein phosphatase, a reflection of the

presence and relative strength of the environmental signals that act through each.

The ability of many protein kinases and protein phosphatases to target more than

one protein provides a means for an environmental signal to coordinately regulate

multiple metabolic processes. For example, the enzymes 3-hydroxy-3-methylglutaryl-

CoA reductase and acetyl-CoA carboxylasethe rate-controlling enzymes for

cholesterol and fatty acid biosynthesis, respectivelyare phosphorylated and

inactivated by the AMP-activated protein kinase. When this protein kinase is activated

either through phosphorylation by yet another protein kinase or in response to the

binding of its allosteric activator 5-AMP, the two major pathways responsible for thesynthesis of lipids from acetyl-CoA are both inhibited.

INDIVIDUAL REGULATORY EVENTS COMBINE TO FORM SOPHISTICATED

CONTROL NETWORKS

Cells carry out a complex array of metabolic processes that must be regulated in

response to a broad spectrum of environmental factors. Hence, interconvertible

enzymes and the enzymes responsible for their interconvesion do not act as isolated

on and off switches. In order to meet the demands of maintaining homeostasis,

these building blocks are linked to form integrated regulatory networks.

One well-studied example of such a network is the eukaryotic cell cycle thatcontrols cell division. Upon emergence from the G0or quiescent state, the extremely

complex process of cell division proceeds through a series of specific phases

designated G1, S, G2, and M (Figure 98).Elaborate monitoring systems, called

checkpoints, assess key indicators of progress to ensure that no phase of the cycle is

initiated until the prior phase is complete.Figure 98outlines, in simplified form, part

of the checkpoint that controls the initiation of DNA replication, called the S phase. A
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protein kinase called ATM is associated with the genome. If the DNA contains a

double-stranded break, the resulting change in the conformation of the chromatin

activates ATM. Upon activation, one subunit of the activated ATM dimer dissociates

and initiates a series, or cascade, of protein phosphorylationdephosphorylation

events mediated by the CHK1 and CHK2 protein kinases, the Cdc25 protein

phosphatase, and finally a complex between a cyclin and a cyclin-dependent proteinkinase, or Cdk. Activation of the Cdk-cyclin complex blocks the G1to S transition,

thus preventing the replication of damaged DNA. Failure at this checkpoint can lead

to mutations in DNA that may lead to cancer or other diseases. Each step in the

cascade provides a conduit for monitoring additional indicators of cell status prior to

entering S phase.

FIGURE 98 A simplified representation of the G1to S checkpoint of the

eukaryotic cell cycle.The circle shows the various stages in the eukaryotic cell cycle.

The genome is replicated during S phase, while the two copies of the genome aresegregated and cell division occurs during M phase. Each of these phases is separated

by a G, or growth, phase characterized by an increase in cell size and the

accumulation of the precursors required for the assembly of the large macromolecular

complexes formed during S and M phases.

SUMMARY


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Homeostasis involves maintaining a relatively constant intracellular and intra-

organ environment despite wide fluctuations in the external environment. This is

achieved via appropriate changes in the rates of biochemical reactions in response

to physiologic need.

The substrates for most enzymes are usually present at a concentration close to

theirKm. This facilitates passive control of the rates of product formation in

response to changes in levels of metabolic intermediates.

Active control of metabolite flux involves changes in the concentration, catalytic

activity, or both of an enzyme that catalyzes a committed, rate-limiting reaction.

Selective proteolysis of catalytically inactive proenzymes initiates conformational

changes that form the active site. Secretion as an inactive proenzyme facilitates

rapid mobilization of activity in response to injury or physiologic need and may

protect the tissue of origin (eg, autodigestion by proteases).

Binding of metabolites and second messengers to sites distinct from the catalytic

site of enzymes triggers conformational changes that alter VmaxorKm.

Phosphorylation by protein kinases of specific seryl, threonyl, or tyrosyl

residuesand subsequent dephosphorylation by protein phosphatasesregulates

the activity of many human enzymes. The protein kinases and phosphatases that

participate in regulatory cascades that respond to hormonal or second messenger

signals constitute regulatory networks that can process and integrate complex

environmental information to produce an appropriate and comprehensive cellular

response.

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Elgin SC, Reuter G. In Allis CD, Jenuwein T, Reinberg D, et al (editors):

Epigenetics, Cold Spring Harbor Laboratory Press, 2007.

Johnson LN, Lewis RJ: Structural basis for control by phosphorylation. Chem Rev

2001;101:2209.

Muoio DM, Newgard CB: Obseity-related derangements in metabolic regulation.

Anu Rev Biochem 2006;75:403.

Scriver CR, Sly WS, Childs B, et al (editors): The Metabolic and Molecular Bases ofInherited Disease,8th ed. McGraw-Hill, 2000.

Stieglitz K, Stec B, Baker DP, et al: Monitoring the transition from the T to the R

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Tu BP, Kudlicki A, Rowicka M, et al: Logic of the yeast metabolic cycle: temporal

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