Denu J M, Dixon J E
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5910-4. doi: 10.1073/pnas.92.13.5910.
Dual-specific protein-tyrosine phosphatases have the common active-site sequence motif HCXXGXXRS(T). The role of the conserved hydroxyl was investigated by changing serine-131 to an alanine (S131A) in the dual-specific protein-tyrosine phosphatase VHR. The pH profile of the kcat/Km value for the S131A mutant is indistinguishable from that of the native enzyme. In contrast, the kcat value for S131A mutant is 100-fold lower than that for the native enzyme, and the shape of the pH profile was perturbed from bell-shaped in the native enzyme to a pH-independent curve over the pH range 4.5-9.0. This evidence, along with results from a previous study, suggests that the S131A mutation alters the rate-limiting step in the catalytic mechanism. Formation of a phosphoenzyme intermediate appears to be rate-limiting with the native enzyme, whereas in the S131A mutant breakdown of the intermediate is rate-limiting. This was confirmed by the appearance of a burst of p-nitrophenol formation when p-nitrophenyl phosphate rapidly reacted with the S131A enzyme in a stopped-flow spectrophotometer. Loss of this hydroxyl group at the active site dramatically diminished the ability of the enzyme to hydrolyze the thiol-phosphate intermediate without exerting any significant change in the steps leading to and including the formation of the intermediate. Consistent with rate-limiting intermediate formation in the native enzyme, the rate of burst in the S131A mutant was 1.5 s-1, which agrees well with the kcat value of 5 s-1 observed for native enzyme. The amplitude of the burst was stoichiometric with final enzyme concentration, and the slow linear rate (0.06 s-1) of p-nitrophenol formation after the burst was in agreement with the steady-state determined value of kcat (0.055 s-1).
双特异性蛋白酪氨酸磷酸酶具有共同的活性位点序列基序HCXXGXXRS(T)。通过将双特异性蛋白酪氨酸磷酸酶VHR中的丝氨酸-131替换为丙氨酸（S131A），研究了保守羟基的作用。S131A突变体的kcat/Km值的pH曲线与天然酶的pH曲线没有区别。相比之下，S131A突变体的kcat值比天然酶低100倍，并且pH曲线的形状从天然酶中的钟形在4.5-9.0的pH范围内扰动为与pH无关的曲线。这一证据以及先前研究的结果表明，S131A突变改变了催化机制中的限速步骤。天然酶中磷酸酶中间体的形成似乎是限速步骤，而在S131A突变体中，中间体的分解是限速步骤。当对硝基苯磷酸酯在停流分光光度计中与S131A酶快速反应时，对硝基苯酚形成的爆发现象证实了这一点。活性位点处该羟基的缺失显著降低了酶水解硫醇-磷酸中间体的能力，而在导致中间体形成并包括中间体形成的步骤中没有产生任何显著变化。与天然酶中限速中间体形成一致，S131A突变体中的爆发速率为1.5 s-1，这与天然酶观察到的5 s-1的kcat值非常吻合。爆发的幅度与最终酶浓度成化学计量关系，爆发后对硝基苯酚形成的缓慢线性速率（0.06 s-1）与kcat的稳态测定值（0.055 s-1）一致。
