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studies on New Reactions in Glycosylase Catalysis

糖基酶催化新反应的研究

基本信息

批准号：
02806014
负责人：
MATSUI Hirokazu
金额：
$ 1.09万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Aspergillus niger alpha-glucosidase catalyzed the slow hydroysis of beta-Dglucopyranosyl fluoride to form alpha-D-glucose. Maltotriose competitively inhibited the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibited the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase.Also alpha-glucosidases from sugar beet and rice catalyzed the hydrolysis of beta-D-glucosyl fluoride to form alpha-D-glucose. The reaction was slow, with V/K 1-2% of that for PNP alpha-D-glucoside hydrolysis, but was not due to any impurity in the substrate or to contaminating beta-glucosidase or glucoamylase. Maltopentaose was a linear competitive inhibitor of PNP alpha-glucoside hydrolysis in each case. Almond beta-glucosidase, promoted alpha-D-glucosyl fluoride hydrolysis to form beta-glucose at an exceedingly low rate. This weak reaction did not stem from any impurity in substance, … More or to contamination with alpha-glucosidase or glucoamylase, but it was partly(ca. 20%)attributable to a trace of accompanying trehalase.That the disfavored D-glucosyl fluoride in each case was converted to a product of the same configuration as from enitols or favored D-glucosyl substrates provides ndw evidence for a separate stage of catalysis in which the steric outcome of reactions of a glucosidase is strictly conserved by protein structure regardless of substrate configuration.Coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and D-galacto-octenitol, each a known substrate for beta-galactosidase. The enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center. The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase. Less

黑曲霉α-葡萄糖苷酶催化β-D-吡喃葡萄糖氟化物缓慢水解生成α-D-葡萄糖。麦芽三糖竞争性地抑制该酶的水解，而β-D-葡萄糖基氟化物则竞争性地抑制对硝基苯基α-D-吡喃葡萄糖苷的水解，这表明β-D-葡萄糖基氟化物与已知的α-葡萄糖苷酶底物结合在同一个位置上。甜菜和大米中的α-葡萄糖苷酶也催化了β-D-葡萄糖基氟化物的水解生成α-D-葡萄糖。反应缓慢，V/K值仅为PNPα-D-葡萄糖苷水解率的1~2%，但不是由于底物中的任何杂质或污染了β-葡萄糖苷酶或糖淀粉酶。在每种情况下，Maltopentaose都是PNPα-葡萄糖苷水解的线性竞争性抑制剂。杏仁β-葡萄糖苷酶以极低的速率促进α-D-葡萄糖基氟化物的水解生成β-葡萄糖。这种弱反应不是由物质中的任何杂质引起的，…更多的原因是被α-葡萄糖苷酶或糖淀粉酶污染，但部分(约20%)可归因于伴随的少量海藻糖酶。在每种情况下，不受欢迎的D-葡萄糖基氟化物从烯醇或有利的D-葡萄糖基底物转化为与相同构型的产物，这为单独的催化阶段提供了新的证据，在该阶段中，葡萄糖苷酶的反应立体结果严格由蛋白质结构保守，而与底物构型无关。咖啡豆α-半乳糖苷酶被发现催化D-半乳糖和D-半乳辛醇的水合，这两种底物都是β-半乳糖苷酶的已知底物。该酶从环平面下方质子化每个底物，就像α-D-半乳糖苷所假定的那样。这些结果明确地指定了酸性催化基团向α-半乳糖苷酶反应中心的方向。根据糖苷酶催化包括“可塑性”质子化阶段和“保守”产品构型阶段的概念，讨论了这些发现。较少