Derst C, Henseling J, Röhm K H
Institut für Physiologische Chemie, Philipps-Universität, Marburg(Lahn), Germany.
Protein Eng. 1992 Dec;5(8):785-9. doi: 10.1093/protein/5.8.785.
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.
位点特异性诱变已被用于探究那些被认为对大肠杆菌天冬酰胺酶II催化作用至关重要的氨基酸残基。苏氨酸12在所有已知的天冬酰胺酶中都是保守的。T12A突变体对L-天冬氨酸β-异羟肟酸的催化常数降至野生型活性的0.04%，而其米氏常数和对尿素变性的稳定性未发生变化。突变酶T12S表现出几乎正常的活性，但底物特异性发生了改变。用丙氨酸取代苏氨酸119导致活性降低90%，而对底物结合没有明显影响。突变酶S122A表现出正常的催化功能，但在尿素溶液中的稳定性受损。这些数据表明，苏氨酸12的羟基直接参与催化作用，可能是通过与过渡态或中间体形成有利的相互作用。相比之下，苏氨酸119和丝氨酸122这两个假定的失活剂DONV的靶点，在功能上不太重要。
