Covalent modification of proteins

Evolution has provided the cell with a repertoire of 20 amino acids to build proteins. The diversity of amino acid side chain properties is enormous, yet many additional functional groups have been selectively chosen to be covalently attached to side chains and this further increases the unique properties of proteins. Diese additional groups play a regulatory role allowing the cell to respond to changing cellular conditions and events. Known covalent modifications of proteins now include phosphorylation, methylation, acetylation, ubi-quitylation, hydroxylation, uridylylation and glycosyl-ation, among many others. Intense study in this field has shown the addition of a phosphate moiety to a protein... 

McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-ZerZ-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration, j. Org. Chem. 1997, 62, 1820-1825. 

K. Mizutani, T. Electronic and structural requirements for metabolic activation of butylated hydroxytoluene analogs to their quinone methides, intermediates responsible for lung toxicity in mice. Biol. Pharm. Bull. 1997, 20, 571-573. (c) McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-rm-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration. J. Org. Chem. 1997, 62, 1820-1825. (d) Reed, M. Thompson, D. C. Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di- m-butyl-4-methylphenol (BHT). Chem. Res. Toxicol. 1997, 10, 1109-1117. (e) Lewis, M. A. Yoerg, D. G. Bolton, J. L. Thompson, J. Alkylation of 2 -deoxynucleosides and DNA by quinone methides derived from 2,6-di- m-butyl-4-methylphenol. Chem. Res. Toxicol. 1996, 9, 1368-1374. 

Mason JR, Feong FC, Plaxco KW, et al. Two-step covalent modification of proteins. Selective labeling of Schiff base-forming sites and selective blockade of the sense of smell in vivo. J. Am. Chem. Soc. 1985 107 6075-6084. 

A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 6.5. Protein denaturation, for example, entails a partial or complete alteration of the protein s three-dimensional shape. This is underlined by the disruption of the intramolecular forces that stabilize a protein s native conformation, namely hydrogen bonding, ionic attractions and hydrophobic interactions (Chapter 2). Covalent modifications of protein structure that can adversely affect its biological activity are summarized below. 

In the mid-1970s ubiquitin was found to be a covalent modifier of proteins [1]. At the time, it was quite surprising to find a protein that covalently modified another protein. Since then, the reversible covalent modification of proteins by other proteins is known to be commonplace and ubiquitin is used to covalently modify hundreds of proteins, often for the purpose of targeting them to the proteasome for degradation. 

NAD glycohydrolases from rat liver nuclei, 66, 151 poly(ADP-ribose) synthetase from rat liver nuclei, 66, 154 poly(ADP-ribose) synthetase from calf thymus, 66, 159 extraction and quantitative determination of larger than tetrameric endogenous polyadenosine diphosphoribose from animal tissues, 66, 165 covalent modification of proteins by metabolites of NAD, 66, 168 coenzyme activity of NAD bound to polymer supports through the adenine moiety, 66, 176 use of differently immobilized nucleotides for binding NAD -dependent dehydrogenases, 66, 192. 

NO mediates its effects by covalent modification of proteins. There are three major effector targets of NO (Figure 19-1) Metalloproteins... 

What similarities and differences can you find in the covalent modification of proteins shown in figures 9.3 and 9.4 ... 

Some of the spectral effects of specific covalent modifications of proteins, i.e., iodination, oxidation, tyrosinase action, etc., will be discussed briefly. Heme proteins, flavoproteins, and other conjugated proteins will not be discussed except as regards studies involving their amino acid components. 

Table 10.1. Common covalent modifications of protein activity...

Covalent modification of proteins is a potent means of controlling the activity of enzymes and other proteins. Phosphorylation is the most common type of reversible covalent modification. Signals can be highly amplified by phosphorylation because a single kinase can act on many target molecules. The regulatory actions of protein kinases are reversed by protein phosphatases, which catalyze the hydrolysis of attached phosphoryl groups. 

Figure 30.2. Covalent Modifications. Covalent modifications. Examples of reversible covalent modifications of proteins (A) phosphorylation, (B) adenylation.

Cholesterol has several functions including involvement in membrane structure, by modulation of membrane fluidity and permeability, serving as a precursor for steroid hormone and bile acid synthesis, in the covalent modification of proteins, and formation of the central nervous system in embryonic development. The latter role of cholesterol was discovered through mutations and pharmacological agents that block cholesterol biosynthesis that occurs in six steps ... 

Mason, J, R., Leong, F.-C., Plaxco, K., Morton, T. H. 1985. "Two-Step Covalent Modification of Proteins. Selective Labelling of Schiff-Base-Forming Sites and Selective Blockade of the Sense of Smell." Journal of the American Chemical Society, 97 6075—6084. 

Sumoylation is a covalent modification of proteins that is related to, but functionally distinct from ubiquitination (review Wilson and Rangasami, 2001). As in ubiquitinylation, sumoylation involves the covalent attachment of a small protein moiety, termed SUMO, to target proteins. The reactions leading to sumoylation of substrate proteins are related to those involved in ubiquitination. El- and E2 like enzymes are responsible for the attachment of the SUMO moiety to lysine residues of the target protein. As compared to ubiquitination, sumolyation is more sequence specific and requires a particular amino acids in the neighbourhood of the lysine to be modified. 

The administration of TNT to laboratory animals leads to the excretion of 4-NHOH-DNT, 2-NH2-DNT, and 4-NH2-DNT in the urine [59], and to the formation of covalent adducts with microsomal liver and kidney proteins, hemoglobin, and other blood proteins [60], The acid hydrolysis of adducts yielded mainly 2-NH2-DNT (2-ADNT) and 4-NH2-DNT (4-ADNT). Incubation of rat liver microsomes with TNT and NADPH under aerobic conditions resulted in the formation of NH2-DNTs and the transient metabolite 4-NHOH-DNT [57], The formation of covalent protein adducts with TNT metabolites was enhanced by the presence of 02 and decreased by GSH. This is consistent with the scheme of the TNT adduct formation with the central role of the nitroso metabolite (NO-DNT) reaction with protein or nonprotein thiols (RSH Equation 9.11) [57], The acid hydrolysis of the sulfinamide adduct (RS(0)-NH-DNT) formed after the rearrangement of the semimercaptal (RS-N(OH)-DNT Equation 9.12) will yield NH2-DNT. The mixture of NHOH-DNTs inhibits bacterial glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase more efficiently than TNT [61]. This was attributed to the covalent modification of protein -SH groups. 

See also Covalent Modifications to Regulate Enzyme Activity, Covalent Modification of Proteins... 

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