Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites.
Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites.
复制标题
人溶酶体 β-葡萄糖苷酶：催化、糖苷配基和疏水结合位点的动力学特征。
DOI：
10.1016/0003-9861(84)90371-0
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发表时间：
1984
影响因子：
3.9
通讯作者：
Desnick,RJ
中科院分区：
文献类型：
--
作者：
Grabowski,GA;Gatt,S;Kruse,J;Desnick,RJ
通过研究不同酶修饰剂的相互作用，确定了人胎盘溶酶体β-葡萄糖苷酶的三个结合位点。带负电荷的脂质，牛磺胆酸盐和磷脂酰丝氨酸，被证明是4-甲基伞形酮基-β-d-葡萄糖苷水解的非竞争性、非必需的激活剂。使用天然底物葡糖基神经酰胺和低浓度的牛磺胆酸盐（<1.8mm）或磷脂酰丝氨酸（0.5mm）观察到类似的结果。然而，较高的浓度导致葡萄糖神经酰胺水解的复杂的部分抑制。增加浓度的磷脂酰丝氨酸避免牛磺胆酸盐的影响，这表明这些化合物竞争酶上的一个共同的结合位点。葡萄糖基鞘氨醇及其N-己基衍生物是使用任一底物的酶活性的有效非竞争性抑制剂。牛磺胆酸盐（或磷脂酰丝氨酸）和葡萄糖鞘氨醇被证明是相互排斥的，表明竞争一个共同的结合位点。相比之下，辛基-和十二烷基-β-葡萄糖苷是葡糖基神经酰胺或4-甲基伞形酮基-β-d-葡萄糖苷水解的线性混合型抑制剂，表明酶上至少有两个结合位点。仅在牛磺胆酸盐或磷脂酰丝氨酸存在下观察到这些烷基β-葡糖苷的抑制作用。两种烷基β-葡糖苷的竞争组分[Ki（斜率）]随烷基链长度的增加而降低，并且不受牛磺胆酸盐或磷脂酰丝氨酸浓度增加的影响。两种烷基β-葡糖苷的非竞争性组分[Ki（截距）]几乎相同，并且随着牛磺胆酸盐或磷脂酰丝氨酸浓度的增加而降低。这些结果表明，带负电荷的脂质和烷基β-葡萄糖苷不是相互排斥的，而是与酶上不同的结合位点相互作用。葡萄糖酸内酯被证明可以保护酶免受催化位点定向共价抑制剂conduritol B环氧化物的抑制，这表明在一个共同的结合位点存在相互作用。在底物存在的情况下，牛磺胆酸盐促进了对甘草内酯或conduritol B环氧化物的抑制。这些研究表明，溶酶体β-葡萄糖苷酶至少有三个结合位点：（i）切割β-葡萄糖苷部分的催化位点，（ii）结合底物和某些抑制剂的酰基或烷基部分的糖苷配基位点，以及（iii）与带负电荷的脂质相互作用并促进酶催化的疏水位点。
Three binding sites on highly purified lysosomal β-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-β-d-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (<1.8 mm) or phosphatidylserine (0.5 mm). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and itsN-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-β-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-β-d-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl β-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki(slope)] for the two alkyl β-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki(intercept)] was nearly identical for both alkyl β-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl β-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B epoxide, indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal β-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the β-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.