Amino acid substitution enzyme preferable substrates

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Computational enzyme-substrate docking studies carried out on a CRED from Sporoholomyces salmonicolor showed that residues Met 242 and Gin 245 were in close proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagenesis of Met 242 or Gin 245 (replacing these residues with all 19 other possible amino acids) and double mutation of both Met 242 and Gin 245 were carried out to enhance the enzyme s enantioselectivity toward the reduction of para-substituted acetophenones. The enantiopreference was inverted from R)- to (S)-configuration for three Gin 245 mutants that were obtained. Preference for the formation of (S)-enantiomeric alcohols was shown for four Met 242 mutant enzymes when compared to the wild-type enzyme Gin 245 and Met 242/Gln 245 double variations inverted the enantiopreference and affected the enantiomeric purity of the product alcohols. Residues 242 and 245 also exerted an effect on the catalytic activity of this GRED [23]. 

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