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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 10.4Covalent Modification Is a Means of Regulating Enzyme Activity

The covalent attachment of another molecule can modify the activity of enzymes and many other proteins. In these instances, a donor molecule provides a functional moiety that modifies the properties of the enzyme. Most modifications are reversible. Phosphorylation and dephosphorylation are the most common but not the only means of covalent modification. Histones—proteins that assist in the packaging of DNA into chromosomes as well as in gene regulation—are rapidly acetylated and deacetylated in vivo (Section 31.3.4). More heavily acetylated histones are associated with genes that are being actively transcribed. The acetyltransferase and deacetylase enzymes are themselves regulated by phosphorylation, showing that the covalent modification of histones may be controlled by the covalent modification of the modifying enzymes.

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Modification is not readily reversible in some cases. Some proteins in signal-transduction pathways, such as Ras and Src (a protein tyrosine kinase), are localized to the cytoplasmic face of the plasma membrane by the irreversible attachment of a lipid group (Section 12.5.3). Fixed in this location, the proteins are better able to receive and transmit information that is being passed along their signaling pathways (Chapter 15). The attachment of ubiquitin, a protein comprising 72 amino acids, is a signal that a protein is to be destroyed, the ultimate means of regulation (Chapter 23). Cyclin, an important protein in cell-cycle regulation, must be ubiquitinated and destroyed before a cell can enter anaphase and proceed through the cell cycle (Table 10.1).

Table 10.1. Common covalent modifications of protein activity.

Table 10.1

Common covalent modifications of protein activity.

Virtually all the metabolic processes that we will examine are regulated in part by covalent modification. Indeed, the allosteric properties of many enzymes are modified by covalent modification. Table 10.1 lists some of the common covalent modifications.

10.4.1. Phosphorylation Is a Highly Effective Means of Regulating the Activities of Target Proteins

The activities of many enzymes, membrane channels, and other target proteins are regulated by phosphorylation, the most prevalent reversible covalent modification. Indeed, we will see this regulatory mechanism in virtually every metabolic process in eukaryotic cells. The enzymes catalyzing phosphorylation reactions are called protein kinases, which constitute one of the largest protein families known, with more than 100 homologous enzymes in yeast and more than 550 in human beings. This multiplicity of enzymes allows regulation to be fine-tuned according to a specific tissue, time, or substrate.

The terminal (γ) phosphoryl group of ATP is transferred to specific serine and threonine residues by one class of protein kinases and to specific tyrosine residues by another.

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The acceptors in protein phosphorylation reactions are located inside cells, where the phosphoryl-group donor ATP is abundant. Proteins that are entirely extracellular are not regulated by reversible phosphorylation. Table 10.2 lists a few of the known protein kinases. Protein phosphatases reverse the effects of kinases by catalyzing the hydrolytic removal of phosphoryl groups attached to proteins.

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Table 10.2. Examples of serine and threonine kinases and their activating signals.

Table 10.2

Examples of serine and threonine kinases and their activating signals.

The unmodified hydroxyl-containing side chain is regenerated and orthophosphate (Pi) is produced.

It is important to note that phosphorylation and dephosphorylation are not the reverse of one another; each is essentially irreversible under physiological conditions. Furthermore, both reactions take place at negligible rates in the absence of enzymes. Thus, phosphorylation of a protein substrate will take place only through the action of a specific protein kinase and at the expense of ATP cleavage, and dephosphorylation will result only through the action of a phosphatase. The rate of cycling between the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases. Note that the net outcome of the two reactions is the hydrolysis of ATP to ADP and Pi, which has a ΔG of -12 kcal mol-1 (-50 kJ mol-1) under cellular conditions (Section 14.1.2). This highly favorable free-energy change ensures that target proteins cycle unidirectionally between unphosphorylated and phosphorylated forms.

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Phosphorylation is a highly effective means of controlling the activity of proteins for structural, thermodynamic, kinetic, and regulatory reasons:


A phosphoryl group adds two negative charges to a modified protein. Electrostatic interactions in the unmodified protein can be disrupted and new electrostatic interactions can be formed. Such structural changes can markedly alter substrate binding and catalytic activity.


A phosphate group can form three or more hydrogen bonds. The tetrahedral geometry of the phosphoryl group makes these hydrogen bonds highly directional, allowing for specific interactions with hydrogen-bond donors.


The free energy of phosphorylation is large. Of the -12 kcal mol-1 (-50 kJ mol-1) provided by ATP, about half is consumed in making phosphorylation irreversible; the other half is conserved in the phosphorylated protein. Recall that a free-energy change of 1.36 kcal mol-1 (5.69 kJ mol-1) corresponds to a factor of 10 in an equilibrium constant (Section 14.1.3). Hence, phosphorylation can change the conformational equilibrium between different functional states by a large factor, of the order of 104.


Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours. The kinetics can be adjusted to meet the timing needs of a physiological process.


Phosphorylation often evokes highly amplified effects. A single activated kinase can phosphorylate hundreds of target proteins in a short interval. Further amplification can take place because the target proteins may be enzymes, each of which can then transform a large number of substrate molecules.


ATP is the cellular energy currency (Chapter 14). The use of this compound as a phosphoryl group donor links the energy status of the cell to the regulation of metabolism.

Protein kinases vary in their degree of specificity. Dedicated protein kinases phosphorylate a single protein or several closely related ones. Multifunctional protein kinases modify many different targets; they have a wide reach and can coordinate diverse processes. Comparisons of amino acid sequences of many phosphorylation sites show that a multifunctional kinase recognizes related sequences. For example, the consensus sequence recognized by protein kinase A is Arg-Arg-X-Ser-Z or Arg-Arg-X-Thr-Z, in which X is a small residue, Z is a large hydrophobic one, and Ser or Thr is the site of phosphorylation. It should be noted that this sequence is not absolutely required. Lysine, for example, can substitute for one of the arginine residues but with some loss of affinity. Short synthetic peptides containing a consensus motif are nearly always phosphorylated by serine- threonine protein kinases. Thus, the primary determinant of specificity is the amino acid sequence surrounding the serine or threonine phosphorylation site. However, distant residues can contribute to specificity. For instance, changes in protein conformation may alter the accessibility of a possible phosphorylation site.

10.4.2. Cyclic AMP Activates Protein Kinase A by Altering the Quaternary Structure

Protein kinases modulate the activity of many proteins, but what leads to the activation of a kinase? Activation is often a multistep process initiated by hormones (Chapter 15). In some cases, the hormones trigger the formation of cyclic AMP, a molecule formed by cyclization of ATP. Cyclic AMP serves as an intracellular messenger in mediating the physiological actions of hormones, as will be discussed in Chapter 15. The striking finding is that most effects of cAMP in eukaryotic cells are achieved through the activation by cAMP of a single protein kinase. This key enzyme is called protein kinase A or PKA. The kinase alters the activities of target proteins by phosphorylating specific serine or threonine residues. As we shall see, PKA provides a clear example of the integration of allosteric regulation and phosphorylation.

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PKA is activated by cAMP concentrations of the order of 10 nM. The activation mechanism is reminiscent of that of aspartate transcarbamoylase. Like that enzyme, PKA in muscle consists of two kinds of subunits: a 49-kd regulatory (R) subunit, which has high affinity for cAMP, and a 38-kd catalytic (C) subunit. In the absence of cAMP, the regulatory and catalytic subunits form an R2C2 complex that is enzymatically inactive (Figure 10.28). The binding of two molecules of cAMP to each of the regulatory subunits leads to the dissociation of R2C2 into an R2 subunit and two C subunits. These free catalytic subunits are then enzymatically active. Thus, the binding of cAMP to the regulatory subunit relieves its inhibition of the catalytic subunit. PKA and most other kinases exist in isozymic forms for finetuning regulation to meet the needs of a specific cell or developmental stage.

Figure 10.28. Regulation of Protein Kinase A.

Figure 10.28

Regulation of Protein Kinase A. The binding of four molecules of cAMP activates protein kinase A by dissociating the inhibited holoenzyme (R2C2) into a regulatory subunit (R2) and two catalytically active subunits (C).

How does the binding of cAMP activate the kinase? Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile, which matches the consensus sequence for phosphorylation except for the presence of alanine in place of serine. In the R2C2 complex, this pseudosubstrate sequence of R occupies the catalytic site of C, thereby preventing the entry of protein substrates (see Figure 10.28). The binding of cAMP to the R chains allosterically moves the pseudosubstrate sequences out of the catalytic sites. The released C chains are then free to bind and phosphorylate substrate proteins.

10.4.3. ATP and the Target Protein Bind to a Deep Cleft in the Catalytic Subunit of Protein Kinase A

Image tree.jpg The three-dimensional structure of the catalytic subunit of PKA containing a bound 20-residue peptide inhibitor was determined by x-ray crystallography. The 350-residue catalytic subunit has two lobes (Figure 10.29). ATP and part of the inhibitor fill a deep cleft between the lobes. The smaller lobe makes many contacts with ATP-Mg2+, whereas the larger lobe binds peptide and contributes the key catalytic residues. Like other kinases (Section 16.1.1), the two lobes move closer to one another on substrate binding; mechanisms for restricting this domain closure provides a means for regulating protein kinase activity. The PKA structure has broad significance because residues 40 to 280 constitute a conserved catalytic core that is common to essentially all known protein kinases. We see here an example of a successful biochemical solution to the problem of protein phosphorylation being employed many times in the course of evolution.

Figure 10.29. Protein Kinase A Bound to an Inhibitor.

Figure 10.29

Protein Kinase A Bound to an Inhibitor. Image mouse.jpg Three-dimensional structure of a complex of the catalytic subunit of protein kinase A and an inhibitor bearing a pseudosubstrate sequence. The inhibitor (yellow) binds in a cleft between the domains of the enzyme. (more...)

The bound peptide in this crystal occupies the active site because it contains the pseudosubstrate sequence Arg-Arg-Asn-Ala-Ile (Figure 10.30). The structure of the complex reveals the basis for the consensus sequence. The guanidinium group of the first arginine residue forms an ion pair with the carboxylate side chain of a glutamate residue (Glu 127) of the enzyme. The second arginine likewise interacts with two other carboxylates. The nonpolar side chain of isoleucine, which matches Z in the consensus sequence, fits snugly in a hydrophobic groove formed by two leucine residues of the enzyme.

Figure 10.30. Binding of Pseudosubstrate to Protein Kinase A.

Figure 10.30

Binding of Pseudosubstrate to Protein Kinase A. The two arginine side chains of the pseudosubstrate form salt bridges with three glutamate carboxylates. Hydrophobic interactions are also important in the recognition of substrate. The isoleucine residue (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22399


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