人酸性β-葡萄糖苷酶。使用千金藤醇B环氧化物衍生物研究具有催化活性的正常和戈谢病酶。
Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.
作者信息
Grabowski G A, Osiecki-Newman K, Dinur T, Fabbro D, Legler G, Gatt S, Desnick R J
出版信息
J Biol Chem. 1986 Jun 25;261(18):8263-9.
Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.
人酸性β-葡萄糖苷酶（葡糖神经酰胺酶；EC 3.2.1.45）可裂解葡糖神经酰胺和合成β-葡萄糖苷的糖苷键。环醇B环氧化物（CBE）及其溴化衍生物是基于机制的抑制剂，它们与酸性β-葡萄糖苷酶的催化位点共价结合。开发了使用溴化氚标记的CBE和单特异性抗人胎盘酸性β-葡萄糖苷酶IgG的方法，以测定来自正常来源的纯胎盘酶制剂中功能性酸性β-葡萄糖苷酶催化位点的摩尔浓度；然后使用具有十二烷酰基（2135±45 min⁻¹）和己酰基（3200±410 min⁻¹）脂肪酸酰基链的葡糖神经酰胺底物以及具有甲基（2235±197 min⁻¹）、庚基（1972±152 min⁻¹）、壬基（2220±247 min⁻¹）和十一烷基（773±44 min⁻¹）烷基链的4-烷基伞形酮基β-葡萄糖苷底物，根据Vmax = [Et]kcat计算kcat值。在正常脾脏粗制剂中，酸性β-葡萄糖苷酶的相应kcat值约为这些值的60%。相比之下，两名1型阿什肯纳兹犹太戈谢病（AJGD）患者的突变脾脏酸性β-葡萄糖苷酶的kcat值降低了约1.5至3倍，并且每种底物的Km值与正常酸性β-葡萄糖苷酶的Km值相似。正常和1型AJGD酶与CBE以1:1化学计量比的相互作用符合在共价失活之前形成可逆EI复合物的模型。对于CBE，正常酶（0.051±0.009 min⁻¹）和1型AJGD酶（0.058±0.016 min⁻¹）的相等kmax值（最大失活速率）与kcat的微小差异一致。相比之下，1型AJGD酶的Ki值（解离常数）（839±64 μM）约为正常Ki值（166±57 μM）的5倍。这些结果表明，具有催化活性的1型AJGD酸性β-葡萄糖苷酶具有近乎正常的水解能力，并表明在酸性β-葡萄糖苷酶活性位点或其附近存在氨基酸取代，导致突变酶活性在体内不稳定。
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