Poyner Russell R, Larsen Todd M, Wong Se-Wei, Reed George H
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53705, USA.
Arch Biochem Biophys. 2002 May 15;401(2):155-63. doi: 10.1016/S0003-9861(02)00024-3.
Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.
晶体学和动力学方法已被用于表征酵母烯醇化酶的一个位点特异性变体，其中活性位点侧翼的丝氨酸39已被替换为丙氨酸。在野生型酶中，丝氨酸39的羰基和羟基螯合第二个二价金属离子等价物，有效地将侧翼固定在完全配位的活性位点上方。以Mg(2+)作为激活阳离子时，如先前报道的那样，S39A烯醇化酶的活性不到野生型活性的0.01%[J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351 - 355]。对(2)H动力学同位素效应的测量表明，从2 - 磷酸甘油酸(2 - PGA)中提取质子是显著的速率决定步骤。对同位素效应的分析提供了有关烯醇化物中间体形成和分解相对速率的信息。此外，用不同种类的二价金属离子进行的测定表明，对于S39A烯醇化酶（与野生型烯醇化酶的情况不同），亲电性更强的金属离子能促进更高的活性。S39A变体的动力学结果支持以下观点：限速产物释放降低了野生型烯醇化酶与亲电性更强的金属离子结合时的活性，并且金属离子用于使2 - PGA的C2 - 质子酸化。S39A烯醇化酶与Mg(2+)和抑制剂膦酰基乙酰氧肟酸(PhAH)共结晶。该结构以2.1 Å的分辨率解析并精制。该结构证实了突变蛋白中活性位点侧翼打开的推测。PhAH与两个Mg离子螯合，如同在野生型复合物的相应结构中一样。催化基团赖氨酸345和谷氨酸211以及“辅助”残基谷氨酸168和赖氨酸396的侧链位置相对于与野生型蛋白形成的复合物几乎没有变化。在野生型复合物中与膦酸酯氧形成氢键的组氨酸159距离最近的膦酸酯氧5.7 Å，并且包含组氨酸159的环(154 - 166)从活性中心移开。一个外围环，谷氨酸251 - 甘氨酸275，也移动以开放对活性位点的通道。
