Protein regulation covalent modification

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

GTP can be a phosphate donor in reactions such as that catalyzed by phosphoenopyruvate carboxykinase during gluconeogenesis. However, ATP is more commonly used. GTP plays important roles as the energy donor for protein synthesis and in allosteric regulation/covalent modification of the G proteins involved in signal transduction. 

See also a-Helix, / -Sheet, Factors Determining Secondary and Tertiary Structure, Thermodynamics of Protein Folding, Dynamics of Protein Folding, Covalent Modifications to Regulate Enzyme Activity (from Chapter 11). 

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

Histone acetylation is a reversible and covalent modification of histone proteins introduced at the e-amino groups of lysine residues. Histones and DNA form a complex - chromatin - which condenses DNA and controls gene activity. Current models interpret histone acetylation as a means to regulate chromatin activity. 

Small tfbiquitin-like modifier represents a family of evolutionary conserved proteins that are distantly related in amino-acid sequence to ubiquitin, but share the same structural folding with ubiquitin proteins. SUMO proteins are covalently conjugated to protein substrates by an isopeptide bond through their carboxyl termini. SUMO addition to lysine residues of target proteins, termed SUMOylation, mediates post-transla-tional modification and requires a set of enzymes that are distinct from those that act on ubiquitin. SUMOylation regulates the activity of a variety of tar get proteins including transcription factors. 

Mammalian proteins are the targets of a wide range of covalent modification processes. Modifications such as glycosylation, hydroxylation, and fatty acid acylation introduce new 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, adenylylation), the most common by far is phosphorylation-dephos-phorylation. Protein kinases phosphorylate proteins by... 

Many proteins can be phosphorylated at multiple sites or are subject to regulation both by phosphorylation-dephosphorylation and by the binding of allosteric ligands. Phosphorylation-dephosphorylation 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 between active and inactive forms by the binding of second messengers or by covalent modification by phosphorylation-dephosphorylation. 

Figure 1 Covalent modifications of DNA and histones play a fundamental role in the regulation of differentiation and development. The writers, readers, and erasers of this dynamic code are potentially amenable to modulation with small molecules. Lysine methylation is a critical posttranslational modification influencing chromatin function (PMT = protein lysine methyltransferase, royal family proteins bind KMe, KDM = lysine demethylase).

Allosteric regulators bind to the target enzyme in a non-covalent manner. An entirely different enzyme control mechanism is covalent modification. Here, the conformation of the enzyme protein, and thereby its activity, is changed by the... 

The change in the conformation of the control enzyme brought about by covalent modification alters the activity of the control enzyme and so regulates substrate flux through that step. This fact underlines the importance of the three-dimensional structure of an enzyme. The inclusion of phosphates may bring about quite a small architectural change to the protein structure but it is sufficient to affect substrate binding and therefore enzyme activity. 

Figure 20.31 The principle of interconversion cycles in regulation of protein activity or changes in protein concentration as exemplified by translation/proteolysis or protein kinase/protein phosphatase. They result in very marked relative changes in regulator concentration or enzyme activity. The significance of the relative changes (or sensitivity in regulation) is discussed in Chapter 3. The principle of regulation by covalent modihcation is also described in Chapter 3. The modifications in cyclin concentration are achieved via translation and proteolysis, which, in effect, is an interconversion cycle. For the enzyme, they are achieved via phosphorylation and dephosphorylation reactions. In both cases, the relative change in concentration/activity by the covalent modification is enormous. This ensures, for example, that a sufficient increase in cyclin can occur so that an inactive cell cycle kinase can be converted to an active cell cycle kinase, or that a cell cycle kinase can be completely inactivated. Appreciation of the common principles in biochemistry helps in the understanding of what otherwise can appear to be complex phenomena.

C. One of the major mechanisms for regulation of preexisting enzymes is via covalent modification, usually by protein phosphorylation or dephosphorylation. 

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