title: Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.
abstract: Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.
authors.affiliation: Institut für Physiologische Chemie, Philipps-Universität, Marburg(Lahn), Germany.
authors.forename: C
authors.initials: C
authors.lastname: Derst
authors.forename: J
authors.initials: J
authors.lastname: Henseling
authors.forename: K H
authors.initials: KH
authors.lastname: Röhm
mesh_terms: D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013329:Structure-Activity Relationship; D013912:Threonine
chemical_list: D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D001215:Asparaginase