Kinetic studies on the broad-specificity β-D-glucosidase from pig kidney
Kinetic studies on the broad-specificity β-D-glucosidase from pig kidney
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猪肾广泛特异性β-D-葡萄糖苷酶的动力学研究
DOI：
10.1042/bj2560139
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发表时间：
1988
影响因子：
4.1
通讯作者：
L. Kiss
中科院分区：
文献类型：
--
作者：
I. Pócsi;L. Kiss
从猪肾皮质中分离出一种广谱的β - d-葡萄糖苷酶，并通过快速纯化程序纯化到均质性。pI（5.14 +/- 0.05）、Mr（59,000 +/- 2000）和对硝基苯糖苷（半乳糖糖苷、葡萄糖苷、阿拉伯糖苷、木pyrano苷）的比活性与先前发表的其他来源和器官的细胞质β - d -葡萄糖苷酶的比活性相当。混合底物实验和葡萄糖-(1----5)-内酯的抑制研究表明，包含一个催化位点和一个糖结合位点的单一活性中心负责所有四种合成底物的分裂。用底物类似物进行的抑制实验表明：(i)糖苷的主要结合决定因素是糖苷元部分，（ii）酶的阴离子侧链（可能是羧基）与糖苷键相互作用，（iii）糖苷元的性质显著影响碳水化合物部分的结合。对亚硝基苯基衍生物的抑制常数与相应底物的Km值吻合较好。因此，Michaelis常数可以看作是真正的平衡常数（Ks）。Daniels[1983年匹兹堡大学博士论文]为类似的肝酶提出的底物分裂的“三点附着模型”也适用于猪肾中的β - d -葡萄糖苷酶。讨论了“附件”的可能性质。
: A broad-specificity beta-D-glucosidase from pig kidney cortex was isolated and purified to homogeneity by a rapid purification procedure. The pI (5.14 +/- 0.05), Mr (59,000 +/- 2000) and specific activities with several p-nitrophenyl glycosides (galactopyranoside, glucopyranoside, arabinopyranoside, xylopyranoside) were comparable with those published previously for cytoplasmic beta-D-glucosidase from other sources and organs. Mixed-substrate experiments and inhibition studies with glucono-(1----5)-lactone revealed that a single active centre, containing one catalytic site and one saccharide-binding site, was responsible for the splitting of all four synthetic substrates. Inhibition experiments with substrate analogues demonstrated that (i) the major binding determinant of the glycosides was the aglycone moiety, (ii) an anionic side chain of the enzyme (probably a carboxy group) interacted with the glycosidic linkages and (iii) the properties of the aglycone significantly influenced the binding of the carbohydrate moiety. The inhibition constants of the p-nitrothiophenyl derivatives were in good agreement with the Km values of the corresponding substrates. Therefore the Michaelis constants could be regarded as true equilibrium constants (Ks). The 'three-point-attachment model' of the substrate splitting, proposed by Daniels [(1983) Ph.D. Dissertation, University of Pittsburgh] for the analogous liver enzyme, was applicable for beta-D-glucosidase from pig kidney too. The possible nature of the 'attachments' is discussed.