ELECTROPHILIC CATALYSIS IN TRIOSEPHOSPHATE ISOMERASE - THE ROLE OF HISTIDINE-95
ELECTROPHILIC CATALYSIS IN TRIOSEPHOSPHATE ISOMERASE - THE ROLE OF HISTIDINE-95
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DOI：
10.1021/bi00226a005
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发表时间：
1991-03-26
期刊：
影响因子：
2.9
通讯作者：
KNOWLES, JR
中科院分区：
文献类型：
--
作者：
KOMIVES, EA;CHANG, LC;KNOWLES, JR
用傅里叶变换红外光谱和X射线晶体学研究了组氨酸-95对磷酸丙糖异构酶的亲电催化作用。 已知与野生型酶结合的磷酸二羟丙酮的羰基伸缩频率（在1713 cm-1）比溶液中游离的磷酸二羟丙酮的羰基伸缩频率（在1732 cm-1）低19 cm-1，并且这种伸缩频率的降低归因于酶促亲电体，其使底物羰基朝向烯醇化的过渡态极化。 红外光谱的底物结合到两个定点突变的酵母磷酸丙糖异构酶，其中组氨酸-95已被改变为谷氨酰胺或天冬酰胺显示未受干扰的羰基伸缩频率之间的1732和1742 cm-1。 当组氨酸-95被去除时，羰基极化的缺乏表明组氨酸-95确实是催化亲电试剂，至少对于磷酸二羟丙酮。 谷氨酰胺突变体（H95 Q）的动力学研究表明，该酶遵循一个微妙的不同的质子转移机制，只涉及一个单一的酸碱催化基团。 这些发现表明组氨酸-95在野生型酶中作为一般酸碱催化剂的额外作用。 H95 Q突变体的X-射线晶体结构与中间类似物，phosphoglycolohyxamate，结合在活性位点已被解决到2.8埃的分辨率，这个结构清楚地暗示谷氨酸-165，在野生型异构酶的催化碱，作为唯一的酸-碱催化剂的突变酶。 谷氨酸-165是H95 Q突变体蛋白中唯一显著移位的残基：羧酸基团从其在野生型酶中的位置移动了2埃以上。 谷氨酸-165的重新定位导致更接近底物的羧酸氧与底物的C-1和C-2以及O-1和O-2几乎等距。 事实上，谷氨酸-165被募集来进行质子转移，涉及底物氧在催化过程中的H95 Q酶强烈表明，组氨酸-95是负责这种功能的野生型蛋白质。 因此，组氨酸-95在磷酸丙糖异构化的催化中起着亲电试剂和一般酸碱的双重作用。
Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-angstrom resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme. Glutamate-165 is the only residue that is significantly displaced in the H95Q mutant protein: the carboxylate group has moved by more than 2 angstrom from its position in the wild-type enzyme. The repositioning of glutamate-165 causes the carboxylate oxygen that is closer to the substrate to be almost equidistant from both C-1 and C-2 and both O-1 and O-2 of the substrate. The fact that glutamate-165 is recruited to perform the proton transfers involving the substrate oxygens during catalysis by the H95Q enzyme strongly suggests that histidine-95 is responsible for this function in the wild-type protein. Histidine-95 thus plays a dual role as an electrophile and as a general acid-base in the catalysis of triose phosphate isomerization.