Amino acid residues chemical modification sites

Asp-49/35 is essential to optimal calcium binding and catalysis. Replacement of Asp-49 with other amino acids or chemical modification of the side-chain carboxylate reduces Acat to less than 5% of native rates (Fleer etai, 1981 Van den Berghe/a/., 1989). The close spatial coupling of Asp-49/35 with its respective catalytic histidine (His-48/34) ensures a fixed active site geometry. Stability of this bihelical substructure is critical because His-48/34 is supported by a side chain from an adjacent segment of the same helix (Tyr-52) and must hydrogen bond with a residue from the opposed helix (Asp-99/64) for function. 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

Group-specific chemical modification remains a useful method for studies of structure-function relationships in protein molecules, although unambiguous identification of essential amino acid residues and elucidation of their function are nowadays accomplished mainly by X-ray crystallography and site-directed mutagenesis. Chemical modifications... 

Even when the purpose of a given posttranslational modification is understood, an examination of how and where it occurs is also likely to yield only limited information. The cell biological sites and processes involved in the reactions, the specificity by which certain amino acid residues or specific peptide bonds are selected for chemical modification and the mechanism by which the transformations are carried out remain obscure for a large number of these reactions. 

Ideally, chemical modification should be specific for only one type of amino acid residue, and should have minimal effects on the structure and function of the protein. These ideals are met only rarely, and consequently there are limitations to the conclusions that can be drawn from chemical modification experiments. Table 5-6 lists several reagents in common use. Some of the most popular of these, which are used in chemical modification of residues in the active sites of enzymes, are considered more fully below. 

All these bioelectrocatalytic functions of redox proteins are based on the control and enhancement of the electrical communication between the redox sites of the proteins and the electrode support. This is accomplished by the nano-engineering of the surfaces with covalently anchored proteins, the structural aligmnent of the proteins on the electrodes and the chemical modification of the proteins with redox-active units. Preliminary results suggest that two approaches will play important roles in the future development of bioelectronic systems (i) protein mutagenesis with specific functional amino acid residues that can align the protein on the electrode surface and control the electrical contact with the electrode (ii) the synthesis of de novo proteins with tailored bioelectronic and electrobiocatalytic functions. 

The site-specific modification of native proteins is not one of the routine procedures in protein chemistry. It cannot be placed in the same category as end-group labelling or determination of amino acid composition and sequence. The specific chemical modification of a native protein can never be guaranteed because the reactivity of amino acids in a native protein is rarely predictable even if the three-dimensional structure of the protein is known. Unusual pK s of side chains, steric and solvent effects and the proximity of the amino acid residue to a ligand-binding site all influence its reactivity, frequently in opposite directions. However certain well-defined avenues of in-... 

---