Zhang S, McCarter J D, Okamura-Oho Y, Yaghi F, Hinek A, Withers S G, Callahan J W
Division of Neurosciences, Hospital for Sick Children, Toronto, ON, Canada.
Biochem J. 1994 Nov 15;304 ( Pt 1)(Pt 1):281-8. doi: 10.1042/bj3040281.
Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.
用人溶酶体β-半乳糖苷酶的cDNA永久转染的中国仓鼠卵巢细胞克隆，将酶前体分泌到细胞培养基中，通过亲和层析一步将其纯化至表观均一性。纯化的前体具有完全活性，显示出与成熟胎盘酶相同的最适pH值和Km值，并且具有完整的C末端。完整的酶在Sephacryl S-200分子筛柱上进行层析时，以105,500 Da的单体形式洗脱，而在SDS/PAGE凝胶上，该多肽以88 kDa的多肽形式迁移。用糖肽-N-聚糖酶消化的时间进程表明，前体从88 kDa的蛋白质逐渐转化为72 kDa的蛋白质，这表明该蛋白质中存在五个N-连接的寡糖。前体很容易以甘露糖-6-磷酸依赖的方式被摄取到β-半乳糖苷酶缺陷的GM1-神经节苷脂病成纤维细胞中，并且酶活性恢复到正常水平。我们表明，酶促水解的立体化学过程涉及异头中心β-构型的保留，这表明是双置换机制。此外，在基于机制的失活剂2,4-二硝基苯基-2-脱氧-2-氟-β-D-吡喃半乳糖苷存在下，该酶迅速且不可逆地失活，这意味着存在共价中间体。该酶也被1-乙基-3(3-二甲基氨基丙基)碳二亚胺和苯乙二醛失活，这分别表明活性位点中存在羧酸盐和精氨酸残基。我们得出结论，β-半乳糖苷酶前体在功能上与该酶的成熟溶酶体形式相同，并且是研究该蛋白质结构-功能关系的极好酶源。
