title: Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.
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, beta-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.
authors.affiliation: Rockefeller University, New York, New York, USA.
authors.forename: P W
authors.initials: PW
authors.lastname: Van Ophem
authors.forename: M A
authors.initials: MA
authors.lastname: Pospischil
authors.forename: D
authors.initials: D
authors.lastname: Ringe
authors.forename: D
authors.initials: D
authors.lastname: Peisach
authors.forename: G
authors.initials: G
authors.lastname: Petsko
authors.forename: K
authors.initials: K
authors.lastname: Soda
authors.forename: J M
authors.initials: JM
authors.lastname: Manning
mesh_terms: D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D051263:D-Alanine Transaminase; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014508:Urea; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D006146:Guanidines; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D015091:beta-Alanine; C006977:3-chloroalanine; D011732:Pyridoxal Phosphate; D014508:Urea; D000637:Transaminases; D051263:D-Alanine Transaminase; D019791:Guanidine; D000409:Alanine