Protein side-chain reactivities

Enzymes that perform oxidation-reduction catalysis generally require some type of cofactor to store electrons (and protons) during the catalytic cycle. The cofactors are usually low-molecular weight species that reversibly bind to the enzyme, but in some cases are intrinsic elements of the protein structure. For example, quinoenzymes contain covalently bound quinones derived from tyrosine or tryptophan residues in the protein. These quinocofactors represent a striking extension of protein side-chain reactivity. " ... 

Most reactive metabolites produced by CYP metabolic activation are electrophilic in nature, which means that they can react easily with the nucleophiles present in the protein side chains. Several functional groups are recurrent structural features in M Bis. These groups have been reviewed by Fontana et al. [26] and can be summarized as follows terminal (co or co — 1) acetylenes, olefins, furans and thiophenes, epoxides, dichloro- and trichloroethylenes, secondary amines, benzodioxoles (methylenediox-yphenyl, MDP), conjugated structures, hydrazines, isothiocyanates, thioamides, dithiocarbamates and, in general, Michael acceptors (Scheme 11.1). 

There may be common themes in the role of protein-coenzyme contacts in these B -dependent enzymatic processes. In particular, these contacts could alter the relative stability of the Co(III)—R, Co(II), and Co(I) states to enhance reactivity. For coenzyme B 12-dependent enzymes, the deoxyadenosyl radical generates a substrate-derived radical, either directly or via a radical chain mechanism through the intermediacy of a protein-side-chain-based radical, such as S of cysteine or O of tyrosine. This protein-bound substrate-derived radical then undergoes rearrangement, possibly assisted by protein contacts. Thus, cofactor-protein contacts are probably very important in the activation of the Co—C bond, in altering the Co redox potentials, and in assisting in the rearrangements. 

Figure 5. Transformation of reactive protein side chains to lysinoalanine side chains via elimination and crosslinking formation.

Duewel, H. S., Daub, E., Robinson, V. and Honek, J. F. (2001) Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein. Biochemistry, 40(44), 13167-13176. 

In 1958 Frederick Sanger was awarded his first Nobel Prize in Chemistry for his studies on insulin [1], He had determined its complete amino acid sequence, which proved that proteins have definite stractures. Fifty years later, we have dramatically broadened our understanding of proteins and their role in vivo. Starting from amino acid building blocks, complex proteins have evolved to perform a variety of complex catalytic tasks, hi addition to the amino acid s side chain reactivity. Nature often exploits cofactors and/or metal ions to complement its catalytic repertoire. It is estimated that one third of aU enzymes are metalloproteins and that some of the most difficult biological transformations are mediated by these [2],... 

Figure 6. Transformation of a reactive protein side chain to a lysinoalanine side chain via elimination and crosslink formation. Note that the intermediate carbanion has lost the original asymmetry. The carbanion can combine with a proton to regenerate the original amino acid which is then racemic, or undergo an elimination reaction to form dehydroalanine.

The chemical modification of protein is of importance for a number of reasons. It provides derivatives suitable for sequence analysis, identifies the reactive groups in catalytically active sites of an enzyme, enables the binding of protein to a carrier (protein immobilization) and provides changes in protein properties which are important in food processing. In contrast to free amino acids and except for the relatively small number of functional groups on the terminal amino acids, only the functional groups on protein side chains are available for chemical reactions. 

Short chains of amino acid residues are known as di-, tri-, tetrapeptide, and so on, but as the number of residues increases the general names oligopeptide and polypeptide are used. When the number of chains grow to hundreds, the name protein is used. There is no definite point at which the name polypeptide is dropped for protein. Twenty common amino acids appear regularly in peptides and proteins of all species. Each has a distinctive side chain (R in Figure 45.3) varying in size, charge, and chemical reactivity. 

Much of protein engineering concerns attempts to explore the relationship between protein stmcture and function. Proteins are polymers of amino acids (qv), which have general stmcture +H3N—CHR—COO , where R, the amino acid side chain, determines the unique identity and hence the stmcture and reactivity of the amino acid (Fig. 1, Table 1). Formation of a polypeptide or protein from the constituent amino acids involves the condensation of the amino-nitrogen of one residue to the carboxylate-carbon of another residue to form an amide, also called peptide, bond and water. The linear order in which amino acids are linked in the protein is called the primary stmcture of the protein or, more commonly, the amino acid sequence. Only 20 amino acid stmctures are used commonly in the cellular biosynthesis of proteins (qv). 

In contrast to the lability of certain dN adducts formed by the BHT metabolite above, amino acid and protein adducts formed by this metabolite were relatively stable.28,29 The thiol of cysteine reacted most rapidly in accord with its nucleophilic strength and was followed in reactivity by the a-amine common to all amino acids. This type of amine even reacted preferentially over the e-amine of lysine.28 In proteins, however, the e-amine of lysine and thiol of cysteine dominate reaction since the vast majority of a-amino groups are involved in peptide bonds. Other nucleophilic side chains such as the carboxylate of aspartate and glutamate and the imidazole of histidine may react as well, but their adducts are likely to be too labile to detect as suggested by the relative stability of QMs and the leaving group ability of the carboxylate and imidazole groups (see Section 9.2.3). 

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