Tanimizu N, Ueno H, Hayashi R
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
J Biochem. 1998 Aug;124(2):410-6. doi: 10.1093/oxfordjournals.jbchem.a022127.
Phenylalanine120 is a candidate residue juxtaposing catalytic His12 and His119 in ribonuclease A (RNase A). To clarify its role in construction of the catalytic center, Phe120 was replaced by alanine, tryptophan, leucine, or glutamic acid by site-directed mutagenesis. The transphosphorylation and hydrolysis activities of the mutant RNase As, respectively, toward cytidinyl 3',5' adenosine (CpA) and cytidine 2',3' cyclic monophosphate (C>p) were compared with those of the wild type enzyme. The Km values of the two reactions increased markedly with slight changes in the Kcat values. The pKe values of His12 and His119 in the wild type and mutant enzymes, estimated from the pH dependence of the kcat/Km values, showed little change. The rate of carboxymethylation was reduced markedly by the mutations. The Ki values of the phosphate anion as to hydrolysis activity increased only slightly when Phe120 was replaced by leucine, tryptophan, or alanine. These findings suggest that Phe120 participates in the binding of the substrate, juxtaposing His12 and His119, and in stabilizing the transition state intermediate in the hydrolysis reaction. Furthermore, the decreases in the thermal denaturation temperatures of all the mutants, particularly F120E, indicate that Phe120 also helps maintain the conformational stability of RNase A.
苯丙氨酸120是核糖核酸酶A（RNase A）中与催化性组氨酸12和组氨酸119相邻的候选残基。为阐明其在催化中心构建中的作用，通过定点诱变将苯丙氨酸120分别替换为丙氨酸、色氨酸、亮氨酸或谷氨酸。将突变型RNase A分别对胞苷基3',5'腺苷（CpA）和胞苷2',3'环一磷酸（C>p）的转磷酸化和水解活性与野生型酶的活性进行了比较。两个反应的Km值随Kcat值的轻微变化而显著增加。根据kcat/Km值对pH的依赖性估算的野生型和突变型酶中组氨酸12和组氨酸119的pKe值几乎没有变化。突变使羧甲基化速率显著降低。当苯丙氨酸120被亮氨酸、色氨酸或丙氨酸取代时，磷酸根阴离子对水解活性的Ki值仅略有增加。这些发现表明，苯丙氨酸120参与底物的结合，使组氨酸12和组氨酸119并列，并在水解反应中稳定过渡态中间体。此外，所有突变体，特别是F120E的热变性温度降低，表明苯丙氨酸120也有助于维持RNase A的构象稳定性。
