Wang W, Poland B W, Honzatko R B, Fromm H J
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
J Biol Chem. 1995 Jun 2;270(22):13160-3. doi: 10.1074/jbc.270.22.13160.
Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis. The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics. CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable. However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme. Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate. The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305.
通过定点诱变，将大肠杆菌腺苷酸琥珀酸合成酶假定的天冬氨酸结合位点中的三个精氨酸残基替换为亮氨酸。基于十二烷基硫酸钠聚丙烯酰胺凝胶电泳，将突变酶R303L、R304L和R305L纯化至均一，并通过圆二色光谱法和初速率动力学进行表征。圆二色光谱分析表明，在没有底物的情况下，突变体与野生型酶的二级结构没有差异。突变体和野生型酶对GTP和IMP的Km值相当。然而，相对于野生型酶，突变酶对底物天冬氨酸的Km值增加了50至200倍。尽管突变酶的kcat值降低，但变化不如天冬氨酸Km值变化那么显著。腺苷酸琥珀酸合成酶与IMP和MgGDP-1复合物晶体结构中天冬氨酸的建模与诱变结果一致，天冬氨酸的α-和β-羧酸盐位于精氨酸-131、-303和-305的侧链附近。
