Grace M E, Graves P N, Smith F I, Grabowski G A
Division of Medical and Molecular Genetics, Mount Sinai School of Medicine, New York, New York 10029.
J Biol Chem. 1990 Apr 25;265(12):6827-35.
Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid beta-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Comparative studies were conducted with enzymes expressed from natural (spleen and fibroblasts) alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. Mutant cDNAs containing Thr43 to Lys43 (beta-GlcThr43----Lys) and Asp358 to Glu358 (beta-GlcAsp358----Glu) substitutions and two cDNAs containing Ashkenazi Jewish Gaucher disease Type 1 mutations, Arg120 to Gln120 (beta-GlcArg120----Gln) and Asn370 to Ser370 (beta-GlcAsn370----Ser) were expressed and the gene products characterized by enzymatic, immunologic, and inhibitor studies. Genotypes at the acid beta-glucosidase locus in selected Gaucher disease Type 1 patients were determined by allele-specific oligonucleotide hybridization of amplified genomic DNA. Compared with normal, recombinant or natural enzymes expressed from beta-GlcAsn370----Ser alleles had about 2-5-fold decreased specific activity based on CRIM (cross-reacting immunologic material). The beta-GlcArg120----Gln cDNA expressed catalytically inactive CRIM in Sf9; consistent with the 9-fold decreased CRIM-specific activity of the natural enzyme from a beta-GlcArg120----Gln/beta-GlcAsn370----Ser genetic compound. The beta-GlcAsp358----Glu cDNA expressed catalytically inactive CRIM in Sf9 cells. The presence of natural or recombinant enzyme expressed from beta-GlcAsn370----Ser alleles was sufficient to confer 3-5-fold increased IC50 values for deoxynojirimycin, glucosylsphingosine, and N-alkyl-glucosylamine derivatives. Progress curves for inhibition by the slow-tight binding N-alkyl-glucosylamines indicated that the beta-Glc-Asn370----Ser mutation did not alter a conformational change induced by these reaction intermediate analogues. These results provide evidence that the beta-GlcArg120----Gln and beta-GlcAsn370----Ser mutations found in Gaucher disease Type 1 patient genomes are the molecular bases of the enzymatic dysfunction. In addition, the region including Arg120 and that encompassing Asp358 and Asn370 contain residues critical to active site formation or participation in the catalytic mechanism.
为了研究由正常和1型戈谢病等位基因表达的人酸性β-葡萄糖苷酶（EC 3.2.1.45）活性位点功能的分子基础，对其催化特性和抑制剂结合进行了分析。使用杆状病毒表达系统，对从天然（脾脏和成纤维细胞）等位基因或经诱变的cDNA在草地贪夜蛾（Sf9）细胞中表达的酶进行了比较研究。表达了含有苏氨酸43突变为赖氨酸43（β-GlcThr43→Lys）和天冬氨酸358突变为谷氨酸358（β-GlcAsp358→Glu）替代的突变cDNA，以及两个含有阿什肯纳兹犹太1型戈谢病突变的cDNA，精氨酸120突变为谷氨酰胺120（β-GlcArg120→Gln）和天冬酰胺370突变为丝氨酸370（β-GlcAsn370→Ser），并通过酶学、免疫学和抑制剂研究对基因产物进行了表征。通过对扩增的基因组DNA进行等位基因特异性寡核苷酸杂交，确定了选定的1型戈谢病患者酸性β-葡萄糖苷酶基因座的基因型。与正常情况相比，从β-GlcAsn370→Ser等位基因表达的重组或天然酶基于交叉反应免疫物质（CRIM）的比活性降低了约2至5倍。β-GlcArg120→Gln cDNA在Sf9中表达了无催化活性的CRIM；这与来自β-GlcArg120→Gln/β-GlcAsn370→Ser遗传复合物的天然酶的CRIM特异性活性降低9倍一致。β-GlcAsp358→Glu cDNA在Sf9细胞中表达了无催化活性的CRIM。从β-GlcAsn370→Ser等位基因表达的天然或重组酶的存在足以使脱氧野尻霉素、葡萄糖神经酰胺和N-烷基葡萄糖胺衍生物的IC50值增加3至5倍。慢紧密结合的N-烷基葡萄糖胺抑制的进展曲线表明，β-Glc-Asn370→Ser突变不会改变由这些反应中间类似物诱导的构象变化。这些结果提供了证据，表明在1型戈谢病患者基因组中发现的β-GlcArg120→Gln和β-GlcAsn370→Ser突变是酶功能障碍的分子基础。此外，包括精氨酸120的区域以及包含天冬氨酸358和天冬酰胺370的区域含有对活性位点形成或参与催化机制至关重要的残基。
