Kang C, Sun N, Poland B W, Gorrell A, Honzatko R B, Fromm H J
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
J Biol Chem. 1997 May 2;272(18):11881-5. doi: 10.1074/jbc.272.18.11881.
Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp13, Glu14, Lys16, His41, and Arg131, to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the Km of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and Km values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg131 to leucine caused only a 4-fold increase in the Km for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on kcat are consistent with the putative roles assigned to Asp13 (catalytic base), His41 (catalytic acid), and Glu14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg131 stabilizes the conformation of the loop that binds the beta-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTP- and ATP-binding proteins. Crystallographic structures place Lys16 in a position of direct interaction with the gamma-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.
通过定向突变、圆二色光谱法和动力学研究了大肠杆菌腺苷酸琥珀酸合成酶的五个残基（天冬氨酸13、谷氨酸14、赖氨酸16、组氨酸41和精氨酸131）与催化功能和结构组织之间的关系。D13A突变体没有可测量的活性。E14A和H41N突变体表现出野生型酶活性的1%，底物的米氏常数（Km）增加了2至7倍。K16Q突变体具有野生型酶活性的34%，底物的Km值与野生型系统几乎没有变化。相对于野生型酶，精氨酸131突变为亮氨酸仅使天冬氨酸的Km增加了4倍。D13A、E14A和H41N突变对催化常数（kcat）的显著影响与赋予天冬氨酸13（催化碱）、组氨酸41（催化酸）和谷氨酸14（活性位点的结构组织）的假定作用一致。R131L突变对天冬氨酸结合的适度影响也与最近的晶体学研究一致，该研究表明精氨酸131稳定了结合天冬氨酸β-羧酸盐的环的构象。然而，K16Q突变的适度影响与其他GTP和ATP结合蛋白的P环中相应赖氨酸突变引起的显著变化形成对比。晶体学结构将赖氨酸16置于与GTP的γ-磷酸直接相互作用的位置。此外，在腺苷酸琥珀酸合成酶的所有已知序列中，赖氨酸都存在于相应位置。我们认为，赖氨酸16除了在稳定磷酸转移反应的过渡态中起适度作用外，还可能在结构上稳定酶。此外，K16Q突变体催化活性的适度丧失可能给大肠杆菌带来这样的选择性劣势，以至于这种看似无害的突变在自然界中不被容忍。
