KINETIC MECHANISM AND CHARACTERIZATION OF HUMAN BETA-GALACTOSIDASE PRECURSOR SECRETED BY PERMANENTLY TRANSFECTED CHINESE-HAMSTER OVARY CELLS

Chinese hamster ovary cell clones permanently transfected with the cDN A for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneit y in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and K-m values as the ma ture placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elut es as a 105 500 Da monomer, whereas on SDS/PAGE gels the polypeptide m igrates as an 88 kDa polypeptide. A time course of digestion with glyc opeptide-N-glycanase shows the gradual conversion of the precursor fro m 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-deficie nt, GM1-gangliosidosis fibroblasts, and the enzyme activity is returne d to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the ano meric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator ophenyl-2-deoxy-2-fluoro-beta-D-galac topyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylaminopropyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues r espectively in the active site. We conclude that the beta-galactosidas e 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.