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 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.
Zhang
S
S
Division of Neurosciences, Hospital for Sick Children, Toronto, ON, Canada.
McCarter
J D
JD
Okamura-Oho
Y
Y
Yaghi
F
F
Hinek
A
A
Withers
S G
SG
Callahan
J W
JW
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D015342:DNA Probes; D004790:Enzyme Induction; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D010658:Phenylglyoxal / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D015342:DNA Probes; D004792:Enzyme Precursors; D001616:beta-Galactosidase; D010658:Phenylglyoxal