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