Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.

摘要: Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at coenzyme binding site of bacterial D-amino transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement Tyr-31 by Gln (Y31Q) led 99% loss activity. Titration SH groups native Y31Q DTNB proceeded much faster and a greater extent than corresponding titration for wild-type S180A mutant enzymes. The stability each denaturing agents such as urea or guanidine similar, i.e., in their PLP forms, lost 50% activities 5-15% lower concentration did enzyme. Upon removal agent, significant activity restored absence added 5'-phosphate, addition thiols required. In spite its low able form PMP just readily enzymes presence normal substrates. However, beta-chloro-D-alanine better substrate inactivator it enzymes, most likely because formed pyridoxamine 5-phosphate more rapidly other stereochemical fidelity recombinant less racemase conversion L-alanine D-alanine, higher perhaps has flexibility this