Amino acid residue, modification site

Since the modification that causes loss of activity in the enzyme H- K 234 does not interfere with the formation of the PG-PGIP complex, the site re onable for PGIP recognition may redde in a domain different fiom the active e. Studies are now under way to establish wdiich e(s) and amino acid residues of the e/uiiopolygalacturonase are critical for interaction with PGIP. 

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). 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

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. 

The explanation given above implies that stimulation of enzymatic activity by increase of salt concentration is pH dependent and that it will be less pronounced at lower pH values. The dependence of the steady-state reaction rate on salt concentration and pH as shown in Fig. 3 (Zusman, 1990) exactly reflects this prediction. It seems, therefore, at least as a preliminary explanation, that salt concentration affects the p/Ca of Asp-29, which, unlike its counterparts in eDHFR and ZDHFR, has a much lower p a. The question, then, is why the p/Ca of Asp-29 is so different. To answer this question and the validity of the entire model will require elucidation of the detailed kinetic scheme of ADHFR at different salt concentrations, determination of the three-dimensional structure of this enzyme, and modification of the amino acid residues constituting the active site. 

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... 

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