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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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