15.1 What Factors Influence Enzymatic Activity? 453 of enzyme synthesis, are important mechanisms for the regulation of metabolism. By controlling the amount of an enzyme that is present at any moment, cells can ei- ther activate or terminate various metabolic routes. Genetic controls over enzyme levels have a response time ranging from minutes in rapidly dividing bacteria to hours (or longer) in higher eukaryotes. Once synthesized, the enzyme may also be degraded, either through normal turnover of the protein or through specific decay mechanisms that target the enzyme for destruction. These mechanisms are dis- cussed in detail in Chapter 31. Enzyme Activity Can Be Regulated Allosterically Enzymatic activity can also be activated or inhibited through noncovalent interaction of the enzyme with small molecules (metabolites) other than the substrate. This form of control is termed allosteric regulation, because the activator or inhibitor binds to the enzyme at a site other than (allo means “other”) the active site. Further- more, such allosteric regulators, or effector molecules, are often quite different ster- ically from the substrate. Because this form of regulation results simply from re- versible binding of regulatory ligands to the enzyme, the cellular response time can be virtually instantaneous. Enzyme Activity Can Be Regulated Through Covalent Modification Enzymes can be regulated by covalent modification, the reversible covalent attachment of a chemical group. Enzymes susceptible to such regulation are called interconvert- ible enzymes, because they can be reversibly converted between two forms. Thus, a fully active enzyme can be converted into an inactive form simply by the covalent at- tachment of a functional group. For example, protein kinases are enzymes that act in covalent modification by attaching a phosphoryl moiety to target proteins (Figure 15.1). Protein kinases catalyze the ATP-dependent phosphorylation of OOH groups on Ser, Thr, or Tyr side chains. Removal of the phosphate group by a phosphoprotein phosphatase returns the enzyme to its original state. In contrast to the example in the figure, some enzymes exist in an inactive state unless specifically converted into the active form through covalent addition of a functional group. Covalent modification reactions are catalyzed by special converter enzymes, which are themselves subject to metabolic regulation. (Protein kinases are one class of converter enzymes.) Although covalent modification represents a stable alteration of the enzyme, a different con- verter enzyme operates to remove the modification, so when the conditions that fa- vored modification of the enzyme are no longer present, the process can be reversed, restoring the enzyme to its unmodified state. Because covalent modification events are catalyzed by enzymes, they occur very quickly, with response times of seconds or even less for significant changes in metabolic activity. Regulation of Enzyme Activity Also Can Be Accomplished in Other Ways Enzyme regulation is an important matter to cells, and evolution has provided a vari- ety of additional options, including zymogens, isozymes, and modulator proteins. We will discuss these options first and then return to the major topics of this chapter— enzyme regulation through allosteric mechanisms and covalent modification. Enzyme OH Protein phosphatase Protein kinase Enzyme OPO – O – Catalytically inactive, covalently modified form Catalytically active form O H 2 O P i ATP ADP FIGURE 15.1 Enzyme regulation by reversible covalent modification. 454 Chapter 15 Enzyme Regulation Zymogens Are Inactive Precursors of Enzymes Most proteins become fully active as their synthesis is completed and they sponta- neously fold into their native, three-dimensional conformations. Some proteins, how- ever, are synthesized as inactive precursors, called zymogens or proenzymes, that ac- quire full activity only upon specific proteolytic cleavage of one or several of their peptide bonds. Unlike allosteric regulation or covalent modification, zymogen activa- tion by specific proteolysis is an irreversible process. Activation of enzymes and other physiologically important proteins by specific proteolysis is a strategy frequently ex- ploited by biological systems to switch on processes at the appropriate time and place, as the following examples illustrate. Insulin Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin, an important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin (Figure 15.2). Proteolytic Enzymes of the Digestive Tract Enzymes of the digestive tract that serve to hydrolyze dietary proteins are synthesized in the stomach and pancreas as zymogens (Table 15.1). Only upon proteolytic activation are these enzymes able to form a catalytically active substrate-binding site. The activation of chymotrypsino- gen is an interesting example (Figure 15.3). Chymotrypsinogen is a 245-residue polypeptide chain crosslinked by five disulfide bonds. Chymotrypsinogen is con- verted to an enzymatically active form called -chymotrypsin when trypsin cleaves the peptide bond joining Arg 15 and Ile 16 . The enzymatically active -chymotrypsin acts upon other -chymotrypsin molecules, excising two dipeptides: Ser 14 –Arg 15 Proinsulin Val Phe NH 3 1 Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Thr 10 20 30 Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Leu Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Leu Gln Ser Gln Lys Arg Gly 40 50 60 65 Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 1 10 21 COO – S S S S S S Connecting peptide Insulin Val Phe NH 3 1 Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Thr 10 20 30 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 1 10 21 COO – S S S S S S Pro Lys FIGURE 15.2 Proinsulin is an 86-residue precursor to in- sulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of inter- chain disulfide bridges. 2451 Chymotrypsinogen (inactive zymogen) Cleavage at Arg 15 by trypsin 1 -Chymotrypsin (active enzyme) Self-digestion at Leu 13 , Tyr 146 , and Asn 148 by -chymotrypsin Ser Thr Asn 1 ␣-Chymotrypsin (active enzyme) 13 16 146 149 245 Leu Ile Tyr Ala Arg 13 13 245 1481471514 1481471514 14814714 15 ANIMATED FIGURE 15.3 The proteo- lytic activation of chymotrypsinogen. See this figure animated at www.cengage.com/login. Origin Zymogen Active Protease Pancreas Trypsinogen Trypsin Pancreas Chymotrypsinogen Chymotrypsin Pancreas Procarboxypeptidase Carboxypeptidase Pancreas Proelastase Elastase Stomach Pepsinogen Pepsin TABLE 15.1 Pancreatic and Gastric Zymogens 15.1 What Factors Influence Enzymatic Activity? 455 and Thr 147 –Asn 148 . The end product of this processing pathway is the mature pro- tease ␣-chymotrypsin, in which the three peptide chains, A (residues 1 through 13), B (residues 16 through 146), and C (residues 149 through 245), remain together because they are linked by two disulfide bonds, one from A to B and one from B to C. Blood Clotting The formation of blood clots is the result of a series of zymogen activations (Figure 15.4). The amplification achieved by this cascade of enzymatic activations allows blood clotting to occur rapidly in response to injury. Seven of the clotting factors in their active form are serine proteases: kallikrein, XII a , XI a , IX a , VII a , X a , and thrombin. Two routes to blood clot formation exist. The intrinsic pathway is instigated when the blood comes into physical contact with abnormal surfaces caused by injury; the extrinsic pathway is initiated by factors released from injured tissues. The pathways merge at factor X and culminate in clot formation. Thrombin excises peptides rich in negative charge from fibrinogen, converting it to fibrin, a molecule with a different surface charge distribution. Fibrin readily aggre- gates into ordered fibrous arrays that are subsequently stabilized by covalent crosslinks. Thrombin specifically cleaves Arg–Gly peptide bonds and is homologous to trypsin, which is also a serine protease (recall that trypsin acts only at Arg and Lys residues). Isozymes Are Enzymes with Slightly Different Subunits A number of enzymes exist in more than one quaternary form, differing in their relative proportions of structurally equivalent but catalytically distinct polypeptide subunits. A classic example is mammalian lactate dehydrogenase (LDH), which exists as five different isozymes, depending on the tetrameric association of two different subunits, A and B: A 4 , A 3 B, A 2 B 2 , AB 3 , and B 4 (Figure 15.5). The kinetic Intrinsic pathway Damaged tissue surface Kininogen Kallikrein XII XII a XI XI a IX IX a XX a X VIII a VII a VII Tissue factor Trauma Trauma Extrinsic pathway V a II (Prothrombin) II a (Thrombin) I (Fibrinogen) I a (Fibrin) XIII a Crosslinked fibrin clot Final common pathway FIGURE 15.4 The cascade of activation steps leading to blood clotting.The intrinsic and extrinsic pathways con- verge at factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrino- gen into fibrin, which aggregates into ordered filamen- tous arrays that become crosslinked to form the clot. 456 Chapter 15 Enzyme Regulation properties of the various LDH isozymes differ in terms of their relative affinities for the various substrates and their sensitivity to inhibition by product. Different tissues express different isozyme forms, as appropriate to their particular meta- bolic needs. By regulating the relative amounts of A and B subunits they synthe- size, the cells of various tissues control which isozymic forms are likely to assemble and thus which kinetic parameters prevail. 15.2 What Are the General Features of Allosteric Regulation? Allosteric regulation acts to modulate enzymes situated at key steps in metabolic pathways. Consider as an illustration the following pathway, where A is the precur- sor for formation of an end product, F, in a sequence of five enzyme-catalyzed reactions: enz 1 enz 2 enz 3 enz 4 enz 5 A ⎯⎯→ B ⎯⎯→ C ⎯⎯→ D ⎯⎯→ E ⎯⎯→ F In this scheme, F symbolizes an essential metabolite, such as an amino acid or a nu- cleotide. In such systems, F, the essential end product, inhibits enzyme 1, the first step in the pathway. Therefore, when sufficient F is synthesized, it blocks further synthe- sis of itself. This phenomenon is called feedback inhibition or feedback regulation. Regulatory Enzymes Have Certain Exceptional Properties Enzymes such as enzyme 1, which are subject to feedback regulation, represent a distinct class of enzymes, the regulatory enzymes. As a class, these enzymes have cer- tain exceptional properties: 1. Their kinetics do not obey the Michaelis–Menten equation. Their v versus [S] plots yield sigmoid- or S-shaped curves rather than rectangular hyperbolas (Figure 15.6). Such curves suggest a second-order (or higher) relationship between v and [S]; that is, v is proportional to [S] n , where n Ͼ 1. A qualitative description of the mechanism responsible for the S-shaped curves is that binding of one S to a pro- tein molecule makes it easier for additional substrate molecules to bind to the same protein molecule. In the jargon of allostery, substrate binding is cooperative. 2. Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern, and the feedback inhibitor F bears little structural similarity to A, the substrate for the regulatory enzyme. F apparently acts at (a) The five isomers of lactate dehydrogenase A 4 A 3 B A 2 B 2 AB 3 B 4 (b) A 4 A 3 BA 2 B 2 AB 3 B 4 Liver Muscle White cells Brain Red cells Kidney Heart ACTIVE FIGURE 15.5 The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis (see Chapter 18). It needs LDH to regenerate NAD ϩ from NADH so that glycolysis can continue.The lactate produced is released into the blood.The muscle LDH isozyme (A 4 ) works best in the NAD ϩ -regenerating direction. Heart tissue is aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate to fuel the citric acid cycle to obtain energy.The heart LDH isozyme (B 4 ) is inhibited by excess pyruvate so that the fuel won’t be wasted. Test yourself on the con- cepts in this figure at www.cengage.com/login. v [S] Hyperbolic Sigmoid V max FIGURE 15.6 Sigmoid v versus [S] plot.The dotted line represents the hyperbolic plot characteristic of normal Michaelis–Menten-type enzyme kinetics. 15.3 Can Allosteric Regulation Be Explained by Conformational Changes in Proteins? 457 a binding site distinct from the substrate-binding site. The term allosteric is apt, because F is sterically dissimilar and, moreover, acts at a site other than the site for S. Its effect is called allosteric inhibition. 3. Regulatory or allosteric enzymes like enzyme 1 are, in some instances, regulated by activation. That is, whereas some effector molecules such as F exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influ- ences on activity. 4. Allosteric enzymes typically have an oligomeric organization. They are com- posed of more than one polypeptide chain (subunit), and each subunit has a binding site for substrate, as well as a distinct binding site for allosteric effec- tors. Thus, allosteric enzymes typically have more than one S-binding site and more than one effector-binding site per enzyme molecule. 5. The working hypothesis is that, by some means, interaction of an allosteric en- zyme with effectors alters the distribution of conformational possibilities or sub- unit interactions available to the enzyme. That is, the regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind. In addition to enzymes, noncatalytic proteins may exhibit many of these prop- erties; hemoglobin is the classic example. The allosteric properties of hemoglo- bin are the subject of a Special Focus at the end of this chapter. 15.3 Can Allosteric Regulation Be Explained by Conformational Changes in Proteins? The Symmetry Model for Allosteric Regulation Is Based on Two Conformational States for a Protein Various models have been proposed to account for the behavior of allosteric pro- teins. All of them note that proteins can exist in different conformational states. Models usually propose a small number of conformations (two or, at most, three) for a given protein. For example, the model for allosteric behavior of Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux (the MWC model) proposes two conformational states for an allosteric protein: the R (relaxed) state and the T (taut) state. The MWC model is sometimes referred to as the symmetry model be- cause all subunits in an oligomer are assumed to have the same conformation, whether it is R or T. R-state and T-state protein molecules are in equilibrium, with the T conformation greatly favored over the R ([T] ϾϾ [R]), under conditions in which no ligands are present. This model further suggests that substrate and al- losteric activators (positive effectors) bind only to the R state and allosteric in- hibitors (negative effectors) bind only to the T state. Figure 15.7 illustrates such a model for a dimeric protein, each monomer of which has a substrate-binding site and an effector-binding site. Because substrate (S) binds only to the R state, S bind- ing perturbs the R st T equilibrium in favor of more R-state conformers and thus more S binding. That is, S binding is cooperative. The concentration of ligand giv- ing half-maximal response is defined as K 0.5 . (Like K m , the units of K 0.5 are molar- ity; K m cannot be used to describe these constants, because the protein does not conform to the Michaelis–Menten model for enzyme kinetics.) The MWC model accounts for the action of allosteric effectors. Positive effectors bind only to the R state and thus cause a shift of the R st T equilibrium in favor of more R and thus easier S binding. Negative effectors do the opposite; they perturb the R st T equilibrium in favor of T, the conformation that cannot bind S. Note that positive effectors (allosteric activators) cause a decline in the K 0.5 for S (signifying easier binding of S) and negative effectors raise K 0.5 for S (Figure 15.7). Note that the MWC model assumes an equilibrium between conformational states, but ligand binding does not alter the conformation of the protein. 458 Chapter 15 Enzyme Regulation The Sequential Model for Allosteric Regulation Is Based on Ligand-Induced Conformational Changes An alternative model proposed by Daniel Koshland, George Nemethy, and David Filmer (the KNF model) relies on the well-accepted idea that ligand binding trig- gers a change in the conformation of a protein. And, if the protein is oligomeric, ligand-induced conformational changes in one subunit may lead to changes in the conformation of its neighbors. Such ligand-induced conformational change could cause the subunits of an oligomeric protein to shift from a low-affinity state to a high-affinity state. For example, S binding to one monomer may cause the other monomers to adopt conformations with higher affinity for S (Figure 15.8). Inter- estingly, the KNF model also explains how ligand-induced conformational changes could cause subunits of a protein to adopt conformations with little or no affinity for the ligand, a phenomenon referred to as negative cooperativity. The KNF model is termed the sequential model because subunits undergo sequential changes in conformation due to ligand binding. A comparison of the response of velocity to substrate concentration for positive versus negative cooperativity is shown in Figure 15.8c. Changes in the Oligomeric State of a Protein Can Also Give Allosteric Behavior Although the MWC and KNF models are the best-known paradigms for allosteric protein behavior, other models have been put forward. For example, instead of R and T, consider a monomer–oligomer equilibrium for an allosteric protein, where only the oligomer binds S and [monomer] ϾϾ [ oligomer]. This model strongly A dimeric protein that can exist in either of two states: R 0 or T 0 . This protein can bind three ligands: 1) Substrate (S) : Binds only to R at site S 2) Activator (A) : A positive effector that binds only to R at site F 3) Inhibitor (I) : A negative effector that binds only to T at site F 1.0 0.5 0 Y S 01.02.0 +A No A or I +I K 0.5 Effects of A: A + R 0 R 1(A) Increase in number of R-conformers shifts R 0 T 0 so that T 0 R 0 (1) More binding sites for S made available. (2) Decrease in cooperativity of substrate saturation curve. Effects of I: I + T 0 T 1(I) Increase in number of T-conformers (decrease in R 0 as R 0 T 0 to restore equilibrium) Thus, I inhibits association of S and A with R by lowering R 0 level. I increases cooperativity of substrate saturation curve. [S] Substrate R 1(S) Activator R 1(A,S) Activator Substrate R 1(A) T 0 T Inhibitor T 1(I) R 0 RR R ACTIVE FIGURE 15.7 Allosteric effects: A and I binding to R and T, respectively.The linked equilibria lead to changes in the relative amounts of R and T and, therefore, shifts in the substrate saturation curve.The parameters of such a system are that (1) S and A (or I) have different affinities for R and T and (2) A (or I) modifies the apparent K 0.5 for S by shifting the relative R versus T population. Test yourself on the con- cepts in this figure at www.cengage.com/login. 15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes? 459 resembles the MWC model. In yet another model, we might have a monomeric pro- tein with distinct binding sites for several different ligands. In this case, binding of ligand A to its site might cause a conformational change such that the protein shows much greater affinity for S than it would in the absence of A. Or, binding of ligand I might result in a conformational change in the protein such that its affinity for S is abolished. Although the binding of other ligands may affect the affinity of the monomer for S, S binding cannot show cooperativity in monomeric proteins, be- cause, unlike oligomers, the monomer has only one binding site for S. It is important to realize that all of these various models are attempts to use sim- ple concepts to explain the complex behavior of a protein. Although these models provide reasonable approximations and useful insights, the molecular mechanisms underlying allostery cannot be expected to conform rigidly to any one of these mod- els. Shortly, we explore the regulated behavior of a real protein (glycogen phos- phorylase) with these models in mind. 15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes? Covalent Modification Through Reversible Phosphorylation As we saw in Figure 15.1, enzyme activity can be regulated through reversible phos- phorylation; indeed it is the most prominent form of covalent modification in cel- lular regulation. Phosphorylation is accomplished by protein kinases that target spe- cific enzymes for modification. Phosphoprotein phosphatases operate in the reverse direction to remove the phosphate group through hydrolysis of the side- chain phosphoester bond. Because protein kinases and phosphoprotein phos- (a) Binding of S induces a conformational change. Symmetric protein dimer S Asymmetric protein dimer Transmitted conformational change S S If the relative affinities of the various conformations for S are (b) Ͻ р positive cooperativity ensues. If the relative affinities of the various conformations for S are ϽϽ negative cooperativity ensues. S (c) Positive cooperativity No cooperativity Negative cooperativity 0.2 0.4 0.6 V max v 0.8 1.0 3069 K 0.5 [S] FIGURE 15.8 The Koshland–Nemethy–Filmer sequential model for allosteric behavior. (a) S binding can, by induced fit, cause a conformational change in the subunit to which it binds. (b) If subunit interactions are tightly coupled, binding of S to one subunit may cause the other subunit to assume a conformation having a greater or lesser affinity for S.That is, the ligand-induced conformational change in one subunit can affect the adjoining subunit. Such effects could be transmitted between neighboring peptide domains by chang- ing alignments of nonbonded amino acid residues. (c) Theoretical curves for the binding of a ligand to a pro- tein having four identical subunits, each with one binding site for the ligand. The fraction of maximal binding is plotted as a function of [S]/K 0.5 . 460 Chapter 15 Enzyme Regulation phatases work in opposing directions, regulation must be imposed on these con- verter enzymes so that their interconvertible enzyme targets are locked in the de- sired state (active versus inactive) and a wasteful cycle of ATP hydrolysis is avoided. Thus, converter enzymes are themselves the targets of allosteric regulation or cova- lent modification. Protein Kinases: Target Recognition and Intrasteric Control Protein kinases are converter enzymes that catalyze the ATP-dependent phosphoryla- tion of serine, threonine, or tyrosine hydroxyl groups in target proteins (Table 15.2). Phosphorylation introduces a bulky group bearing two negative charges, causing con- formational changes that alter the target protein’s function. (Unlike a phosphoryl group, no amino acid side chain can provide two negative charges.) Protein kinases represent a protein superfamily whose members are widely diverse in terms of size, subunit structure, and subcellular localization. Nevertheless, all share a common cat- alytic mechanism based on a conserved catalytic core/kinase domain of approximately 260 amino acid residues (Figure 15.9). Protein kinases are classified as Ser/Thr and/or Tyr specific. They also differ in terms of the target proteins that they recognize and phosphorylate; target selection depends on the presence of an amino acid se- quence within the target protein that is recognized by the kinase. For example, cAMP- dependent protein kinase (PKA) phosphorylates proteins having Ser or Thr residues within an R(R/K)X(S*/T*) target consensus sequence (* denotes the residue that be- comes phosphorylated). That is, PKA phosphorylates Ser or Thr residues that occur in an Arg-(Arg or Lys)-(any amino acid)-(Ser or Thr) sequence segment (Table 15.2). Targeting of protein kinases to particular consensus sequence elements within pro- teins creates a means to regulate these kinases by intrasteric control. Intrasteric con- trol occurs when a regulatory subunit (or protein domain) has a pseudosubstrate sequence that mimics the target sequence but lacks an OH-bearing side chain at the right place. For example, the cAMP-binding regulatory subunits of PKA (R subunits in Figure 15.10) possess the pseudosubstrate sequence RRGA *I, and this sequence binds to the active site of PKA catalytic subunits, blocking their activity. This pseudo- Protein Kinase Class Target Sequence* Activators I. Ser/Thr protein kinases A. Cyclic nucleotide–dependent cAMP-dependent (PKA) OR(R/K)X(S*/T*)O cAMP cGMP-dependent O(R/K)KKX(S*/T*)O cGMP B. Ca 2ϩ -calmodulin (CaM)–dependent Phosphorylase kinase (PhK) OKRKQIS*VRGLO Phosphorylation by PKA Myosin light-chain kinase (MLCK) OKKRPQRATS*NVO Ca 2ϩ -CaM C. Protein kinase C (PKC) Ca 2ϩ , diacylglycerol D. Mitogen-activated protein kinases OPXX(S*/T*)PO Phosphorylation (MAP kinases) by MAPK kinase E. G-protein–coupled receptors -Adrenergic receptor kinase (BARK) Rhodopsin kinase II. Ser/Thr/Tyr protein kinases MAP kinase kinase (MAPK kinase) OTEYO Phosphorylation by Raf (a protein kinase) III. Tyr protein kinases A. Cytosolic tyrosine kinases (src, fgr, abl, etc.) B. Receptor tyrosine kinases (RTKs) Plasma membrane receptors for hormones such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) *X denotes any amino acid. TABLE 15.2 Classification of Protein Kinases FIGURE 15.9 Protein kinase A is shown complexed with a pseudosubstrate peptide (orange).This complex also includes ATP (red) and two Mn 2ϩ ions (yellow) bound at the active site (pdb id ϭ 1ATP). 15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes? 461 substrate sequence has an alanine residue where serine occurs in the PKA target se- quence; Ala is sterically similar to serine but lacks a phosphorylatable OH group. When these PKA regulatory subunits bind cAMP, they undergo a conformational change and dissociate from the catalytic (C) subunits, and the active site of PKA is free to bind and phosphorylate its targets. In other protein kinases, the pseudosubstrate sequence involved in intrasteric control and the kinase domain are part of the same polypeptide chain. In these cases, binding of an allosteric effector (like cAMP) in- duces a conformational change in the protein that releases the pseudosubstrate se- quence from the active site of the kinase domain. The abundance of many protein kinases in cells is an indication of the great impor- tance of protein phosphorylation in cellular regulation. Exactly 113 protein kinase genes have been recognized in yeast, and 868 putative protein kinase genes have been identified in the human genome. Tyrosine kinases (protein kinases that phosphorylate Tyr residues) occur only in multicellular organisms (yeast has no tyrosine kinases). Tyrosine kinases are components of signaling pathways involved in cell–cell communi- cation (see Chapter 32). Phosphorylation Is Not the Only Form of Covalent Modification That Regulates Protein Function Several hundred different chemical modifications of proteins have been dis- covered thus far, ranging from carboxylation (addition of a carboxyl group), acetylation (addition of an acetyl group, see Figure 29.30), prenylation (see Figure 9.23), and glycosylation (see Figures 7.32–7.39) to covalent attachment of a polypeptide to the protein (addition of ubiquitin to free amino groups on proteins; see Figure 31.8), to name just a few. A compilation of known protein modifications can be found in RESID, the European Bioinformatics Institute online database (http://www.ebi.ac.uk/RESID/). Only a small number of these co- valent modifications are used to achieve metabolic regulation through reversible conversion of an enzyme between active and inactive forms. Table 15.3 presents a few examples. + cAMP cAMP cAMP cAMP cAMP + 2 C C C R R R R R 2 C 2 inactive R 2 –(cAMP) 4 ANIMATED FIGURE 15.10 Cyclic AMP–dependent protein kinase (also known as PKA) is a 150- to 170-kD R 2 C 2 tetramer in mam- malian cells.The two R (regulatory) subunits bind cAMP (K D ϭ 3 ϫ 10 Ϫ8 M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are enzymatically active as monomers. See this figure animated at www.cengage.com/ login. Reaction Amino Acid Side Chain Reaction (see figure indicated) Adenylylation Tyrosine Transfer of AMP from ATP to Tyr-OH (Figure 25.16) Uridylylation Tyrosine Transfer of UMP from UTP to Tyr-OH (Figure 25.17) ADP-ribosylation Arginine Transfer of ADP-ribose from NAD ϩ to Arg (Figure 25.8) Methylation Glutamate Transfer of methyl group from S-adenosylmethionine to Glu ␥-carboxyl group Oxidation-reduction Cysteine (disulfide) Reduction of Cys-S−S-Cys to Cys-SH HS-Cys (Figure 21.27) TABLE 15.3 Additional Examples of Regulation by Covalent Modification 462 Chapter 15 Enzyme Regulation Note that three of these types of covalent modification require nucleoside triphos- phates (ATP, UTP) that are related to cellular energy status; another relies on re- ducing potential within the cell, which also reflects cellular energy status. 15.5 Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification? Glycogen phosphorylase, the enzyme that catalyzes the release of glucose units from glycogen, serves as an excellent example of the many enzymes regulated both by al- losteric controls and by covalent modification. The Glycogen Phosphorylase Reaction Converts Glycogen into Readily Usable Fuel in the Form of Glucose-1-Phosphate The cleavage of glucose units from the nonreducing ends of glycogen molecules is catalyzed by glycogen phosphorylase, an allosteric enzyme. The enzymatic reaction involves phosphorolysis of the bond between C-1 of the departing glucose unit and the glycosidic oxygen, to yield glucose-1-phosphate and a glycogen molecule that is shortened by one residue (Figure 15.11). (Because the reaction involves attack by phosphate instead of H 2 O, it is referred to as a phosphorolysis rather than a hy- drolysis.) Phosphorolysis produces a phosphorylated sugar product, glucose-1-P, which is converted to the glycolytic substrate, glucose-6-P, by phosphoglucomutase (Figure 15.12). In muscle, glucose-6-P proceeds into glycolysis, providing needed energy for muscle contraction. In the liver, hydrolysis of glucose-6-P yields glucose, which is exported to other tissues via the circulatory system. Glycogen Phosphorylase Is a Homodimer Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Figure 15.13). In addition, a regula- CH 2 OH O O CH 2 OH O O CH 2 OH O O CH 2 OH O O + CH 2 OH O CH 2 OH O O CH 2 OH O O CH 2 OH O OOPO 3 2 – OH HO HO OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH HO Nonreducing end residues n n – 1 ␣-D-Glucose-1-phosphate P i residues FIGURE 15.11 The glycogen phosphorylase reaction. HOCH 2 O HH H H HO OH OH OPO 3 2 – H 2– O 3 POCH 2 O HH H H HO OH OH OH H Glucose-1-phosphate Glucose-6-phosphate FIGURE 15.12 The phosphoglucomutase reaction.