10.3 Covalent Modification Is a Means of Regulating Enzyme Activity

The covalent attachment of a molecule to an enzyme or protein can modify its activity. In these instances, a donor molecule provides the functional moiety being attached. Most modifications are reversible. Phosphorylation and dephosphorylation are common means of covalent modification. The attachment of acetyl groups to lysine residues by acetyltransferases and their removal by deacetylases are another example. Histones—proteins that are packaged with DNA into chromosomes—are extensively acetylated and deacetylated in vivo on lysine residues (Section 31.3). More heavily acetylated histones are associated with genes that are being actively transcribed. Although protein acetylation was originally discovered as a modification of histones, we now know that it is a major means of regulation, with more than 2000 different proteins in mammalian cells regulated by acetylation. Protein acetylation appears to be especially important in the regulation of metabolism. The acetyltransferase and deacetylase enzymes are themselves regulated by phosphorylation, showing that the covalent modification of a protein can be controlled by the covalent modification of the modifying enzymes.

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Modification is not readily reversible in some cases. The irreversible attachment of a lipid group causes some proteins in signal-transduction pathways, such as Ras (a GTPase) and Src (a protein tyrosine kinase), to become affixed to the cytoplasmic face of the plasma membrane. Fixed in this location, the proteins are better able to receive and transmit information that is being passed along their signaling pathways (Chapter 14). Mutations in both Ras and Src are seen in a wide array of cancers. The attachment of the small protein ubiquitin can signal that a protein is to be destroyed, the ultimate means of regulation (Chapter 23). The protein cyclin must be ubiquitinated and destroyed before a cell can enter anaphase and proceed through the cell cycle.

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

Modification

Donor molecule

Example of modified protein

Protein function

Phosphorylation

ATP

Glycogen phosphorylase

Glucose homeostasis; energy transduction

Acetylation

Acetyl CoA

Histones

DNA packing; transcription

Myristoylation

Myristoyl CoA

Src

Signal transduction

ADP ribosylation

NAD+

RNA polymerase

Transcription

Farnesylation

Farnesyl pyrophosphate

Ras

Signal transduction

γ-Carboxylation

HCO_3

Thrombin

Blood clotting

Sulfation

3′-Phosphoadenosine-5′-phosphosulfate

Fibrinogen

Blood-clot formation

Ubiquitination

Ubiquitin

Cyclin

Control of cell cycle

Table 10.1: Common covalent modifications of protein activity

Kinases and phosphatases control the extent of protein phosphorylation

We will see phosphorylation used as a regulatory mechanism in virtually every metabolic process in eukaryotic cells. Indeed, as much as 30% of eukaryotic proteins are phosphorylated. The enzymes catalyzing phosphorylation reactions are called protein kinases. These enzymes constitute one of the largest protein families known: there are more than 500 homologous kinases in human beings. This multiplicity of enzymes allows regulation to be fine-tuned according to a specific tissue, time, or substrate.

ATP is the most common donor of phosphoryl groups. The terminal (γ) phosphoryl group of ATP is transferred to a specific amino acid of the acceptor protein or enzyme. In eukaryotes, the acceptor residue is commonly one of the three containing a hydroxyl group in its side chain. Transfers to serine and threonine residues are handled by one class of protein kinases and to tyrosine residues by another. Tyrosine kinases, which are unique to multicellular organisms, play pivotal roles in growth regulation, and mutations in these enzymes are commonly observed in cancer cells.

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Table 10.2 lists a few of the known serine and threonine protein kinases. 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.

Signal

Enzyme

Cyclic nucleotides

Cyclic AMP-dependent protein kinase

Cyclic GMP-dependent protein kinase

Ca2+ and calmodulin

Ca2+−calmodulin protein kinase

Phosphorylase kinase or glycogen synthase kinase 2

AMP

AMP-activated kinase

Diacylglycerol

Protein kinase C

Metabolic intermediates and other “local” effectors

Many target-specific enzymes, such as pyruvate dehydrogenase kinase and branched-chain ketoacid dehydrogenase kinase

Source: Information from D. Fell, Understanding the Control of Metabolism (Portland Press, 1997), Table 7.2.

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

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. However, this sequence is not absolutely required. Lysine, for example, can substitute for one of the arginine residues but with some loss of affinity. 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, a change in protein conformation can open or close access to a possible phosphorylation site.

Protein phosphatases reverse the effects of kinases by catalyzing the removal of phosphoryl groups attached to proteins. The enzyme hydrolyzes the bond attaching the phosphoryl group.

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The unmodified hydroxyl-containing side chain is regenerated and orthophosphate (Pi) is produced. This family of enzymes, of which there are about 200 members in human beings, plays a vital role in cells because the enzymes turn off the signaling pathways that are activated by kinases. One class of highly conserved phosphatase called PP2A suppresses the cancer-promoting activity of certain kinases.

Importantly, the phosphorylation and dephosphorylation reactions 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 take place only through the action of a phosphatase. The result is that target proteins cycle unidirectionally between unphosphorylated and phosphorylated forms. The rate of cycling between the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases.

Phosphorylation is a highly effective means of regulating the activities of target proteins

Phosphorylation is a common covalent modification of proteins in all forms of life, which leads to the question, What makes protein phosphorylation so valuable in regulating protein function that its use is ubiquitous? Phosphorylation is a highly effective means of controlling the activity of proteins for several reasons:

  1. The free energy of phosphorylation is large. Of the −50 kJ mol−1 (−12 kcal mol−1) provided by ATP, about half is consumed in making phosphorylation irreversible; the other half is conserved in the phosphorylated protein. A free-energy change of 5.69 kJ mol−1 (1.36 kcal mol−1) corresponds to a factor of 10 in an equilibrium constant. Hence, phosphorylation can change the conformational equilibrium between different functional states by a large factor, of the order of 104. In essence, the energy expenditure allows for a stark shift from one conformation to another.

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

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

  4. 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.

  5. Phosphorylation often evokes highly amplified effects. A single activated kinase can phosphorylate hundreds of target proteins in a short interval. If the target protein is an enzyme, it can in turn transform a large number of substrate molecules.

  6. ATP is the cellular energy currency (Chapter 15). The use of this compound as a phosphoryl-group donor links the energy status of the cell to the regulation of metabolism.

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Cyclic AMP activates protein kinase A by altering the quaternary structure

Let us examine a specific protein kinase that helps animals cope with stressful situations. The “flight or fight” response is common to many animals presented with a dangerous or exciting situation. Muscle becomes primed for action. This priming is the result of the activity of a particular protein kinase. In this case, the hormone epinephrine (adrenaline) triggers the formation of cyclic AMP (cAMP), an intracellular messenger formed by the cyclization of ATP. Cyclic AMP subsequently activates a key enzyme: protein kinase A (PKA). The kinase alters the activities of target proteins by phosphorylating specific serine or threonine residues. The striking finding is that most effects of cAMP in eukaryotic cells are achieved through the activation of PKA by cAMP.

PKA provides a clear example of the integration of allosteric regulation and phosphorylation. PKA is activated by cAMP concentrations near 10 nM. The quaternary structure is reminiscent of that of ATCase. Like that enzyme, PKA in muscle consists of two kinds of subunits: a 49-kDa regulatory (R) subunit and a 38-kDa catalytic (C) subunit. In the absence of cAMP, the regulatory and catalytic subunits form an R2C2 complex that is enzymatically inactive (Figure 10.16). 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 enzymatically active. Thus, the binding of cAMP to the regulatory subunit relieves its inhibition of the catalytic subunit. PKA, like most other kinases, exists in isozymic forms for fine-tuning regulation to meet the needs of a specific cell or developmental stage. In mammals, four isoforms of the R subunit and three of the C subunit are encoded in the genome.

Figure 10.16: 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). Each R chain includes cAMP-binding domains and a pseudosubstrate sequence.

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 (Figure 10.16). 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. Interestingly, the cAMP-binding domain of the R subunit is highly conserved and found in all organisms.

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ATP and the target protein bind to a deep cleft in the catalytic subunit of protein kinase A

X-ray crystallography reveals the three-dimensional structure of the catalytic subunit of PKA bound to ATP and a 20-residue peptide inhibitor. The 350-residue catalytic subunit of PKA has two lobes, an amino terminal N-lobe and a carboxyl terminal C-lobe (Figure 10.17). ATP and part of the inhibitor fill a deep cleft between the lobes. The N-lobe makes many contacts with ATP–Mn2+, whereas the C-lobe binds the peptide and contributes the key catalytic residues. As with other kinases, the two lobes move closer to one another on substrate binding; mechanisms that restrict this domain closure provide a means of regulating protein kinase activity. The PKA structure has broad significance because residues 40 to 280 constitute a conserved catalytic core, called the kinase fold, that is common to essentially all known protein kinases. We see here an example of a successful biochemical solution to a problem (in this case, protein phosphorylation) being employed many times in the course of evolution.

Figure 10.17: Protein kinase A bound to an inhibitor. This ribbon model shows a complex of the catalytic subunit of protein kinase A with an inhibitor (yellow) bearing a pseudosubstrate sequence. Notice that the inhibitor binds to the active site, a cleft between the domains of the enzyme. The bound ATP (purple)-Mn2+ (green) is in the active site adjacent to the site to which the inhibitor is bound.
[Drawn from 1ATP.pdb.]

The bound peptide in this crystal occupies the active site because it contains the pseudosubstrate sequence Arg-Arg-Asn-Ala-Ile (Figure 10.18). The structure of the complex reveals the interactions by which the enzyme recognizes 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 carboxylate groups. 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 10.18: Binding of pseudosubstrate to protein kinase A. Notice that the inhibitor makes multiple contacts with the enzyme. The two arginine side chains of the pseudosubstrate ionically interact with three glutamate carboxylate groups (green dashed lines). Hydrophobic interactions also are important in the recognition of substrate. The isoleucine residue of the pseudosubstrate is in contact with a pair of leucine residues of the enzyme.

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