REACTION OF BETA-D-GLUCOSIDASE A3 FROM ASPERGILLUS-WENTII WITH D-GLUCAL
REACTION OF BETA-D-GLUCOSIDASE A3 FROM ASPERGILLUS-WENTII WITH D-GLUCAL
复制标题
DOI：
10.1111/j.1432-1033.1979.tb04219.x
复制
发表时间：
1979-01-01
期刊：
影响因子：
--
通讯作者：
ILLIG, HK
中科院分区：
文献类型：
--
作者：
LEGLER, G;ROESER, KR;ILLIG, HK
D-葡萄糖是一种竞争性和非竞争性的混合抑制剂，对文氏链霉菌产生的β-D-葡萄糖苷酶A3有抑制作用，但对底物的稳态水解率接近较慢。无论D-葡萄糖醛直接与底物竞争，还是底物取代在没有底物的情况下预先孵育结合的D-葡萄糖，都能达到相同的速率。竞争抑制的KI和趋于稳定状态的速率常数与D-葡萄糖水合成2-脱氧-D-葡萄糖的动力学常数一致。抑制作用的浓度依赖关系表明，1个D-葡萄糖醛分子以完全抑制的方式结合，但酶-葡萄糖复合体的两相解离动力学和标记研究表明有一个额外的结合部位。在pH为6，温度为0℃时，可分离出一种EI2络合物。C.采用快速离子交换层析。该络合物在室温和pH为4时可以重新激活，所有的D-葡萄糖醛都以2-脱氧-D-葡萄糖的形式释放。在EI2复合体变性后，1个D-葡萄糖醛分子仍与酶结合。(通过放射性多肽的分离和结构测定)该结合部位被鉴定为先前研究中用活性部位定向的抑制剂康杜立醇B环氧化物标记的相同天冬氨酸残基。
D-Glucal acts as a mixed competitive/non-competitive inhibitor against .beta.-D-glucosidase A3 from A. wentii with slow approach to the steady-state rate of substrate hydrolysis. The same rate is reached whether D-glucal competes directly with the substrate or the substrate displaces D-glucal that has been bound by preincubation in the absence of substrate. The Ki for competitive inhibition and the rate constants for the approach to the steady state agree with the kinetic constants for the hydration of D-glucal to 2-deoxy-D-glucose. The concentration dependence of the inhibition shows that 1 molecule of D-glucal binds with complete inhibition but the biphasic dissociation kinetics of the enzyme-glucal complex and labeling studies point to an additional binding site. An EI2 complex can be isolated at pH 6 and 0.degree. C by rapid ion-exchange chromatography. This complex can be reactivated at pH 4 and room temperature; all the D-glucal is released as 2-deoxy-D-glucose. After denaturation of the EI2 complex 1 molecule of D-glucal remains bound to the enzyme. The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active-site-directed inhibitor, conduritol B epoxide, in a previous study.