Liou Benjamin, Grabowski Gregory A
The A. Graeme Mitchell Chair in Human Genetics, Division and Program in Human Genetics, Children's Hospital Medical Center, 3333 Burnet Avenue, ML 4006, Cincinnati, OH 45229-3039, USA.
Mol Genet Metab. 2009 May;97(1):65-74. doi: 10.1016/j.ymgme.2009.01.006. Epub 2009 Feb 13.
The hydrolysis of glucosylceramide by acid beta-glucosidase proceeds via a two-step, double displacement mechanism that includes cleavage of the O-beta-glucosidic bond, enzyme-glucosylation and, then, enzyme-deglucosylation. Two residues that may impact this cycle are N370 and E235. The N370S mutant enzyme is very common in Gaucher disease type 1 patients. Homology and crystal data predictions suggested that E235 is the acid/base catalyst in the hydrolytic reaction. Here, the roles of N370 and E235 in hydrolysis were explored using mutant proteins with selected amino acid substitutions. Heterologously expressed enzymes were characterized using inhibitors, activators, and alternative substrates to gain insight into the effects on the glucosylation (single turnover) and deglucosylation (transglucosylation) steps in catalysis. Specific substitutions at N370 selectively altered only the glucosylation step whereas N370S altered this and the deglucosylation steps. To provide functional data to support E235 as the acid/base catalyst, progress curves with poor substrates with more acidic leaving groups were used in the presence and absence of azide as an exogenous nucleophile. The restoration of E235G activity to nearly wild-type levels was achieved using azide with 2,4-dinitrophenyl-beta-glucoside as substrate. The loss of the acidic arm of the pH optimum activity curve of E235G provided additional functional support for E235 as the acid/base in catalysis. This study provides insight into the function of these residues in acid beta-glucosidase active site function.
酸性β-葡萄糖苷酶对葡萄糖神经酰胺的水解通过两步双取代机制进行，该机制包括O-β-糖苷键的断裂、酶的糖基化，然后是酶的去糖基化。可能影响这一循环的两个残基是N370和E235。N370S突变酶在1型戈谢病患者中非常常见。同源性和晶体数据预测表明E235是水解反应中的酸碱催化剂。在此，使用具有选定氨基酸取代的突变蛋白探索了N370和E235在水解中的作用。使用抑制剂、激活剂和替代底物对异源表达的酶进行表征，以深入了解对催化过程中糖基化（单次周转）和去糖基化（转糖基化）步骤的影响。N370处的特定取代仅选择性地改变了糖基化步骤，而N370S则改变了这一步骤和去糖基化步骤。为了提供功能数据以支持E235作为酸碱催化剂，在有和没有叠氮化物作为外源亲核试剂的情况下，使用了具有更多酸性离去基团的不良底物的进程曲线。使用叠氮化物和2,4-二硝基苯基-β-葡萄糖苷作为底物，E235G的活性恢复到了接近野生型水平。E235G的pH最佳活性曲线酸性臂的丧失为E235作为催化中的酸碱提供了额外的功能支持。这项研究深入了解了这些残基在酸性β-葡萄糖苷酶活性位点功能中的作用。
