Carboxypeptidase Y from Saccharomyces cerevisiae conformational differences reflected in kinetic behaviour in water and deuterium oxide

Abstract

The glycoenzyme carboxypeptidase Y (peptidyl--amnino-acid hydrolase, EC 3.4.16.1), from baker's yeast (British Fermentation Products Strain, Ng 72), of molecular weight 60000, had a protein portion closely similar to those in the literature for carboxypeptidase Y isolations from other yeast sources, but was 25.3 wt% carbohydrate (mannose 83.3% by wt., glucosamine 10.3% by wt. with traces of galactose and galactosamine). Circular dichroic spectra indicated that the enzyme lost its β-structure as the pH was lowered from 8.08 to 4.16. At p 2H 8.22 in 2H 2O media the conformation of this enzyme was different from that observed at pH 8.08. A tyrosine residue appeared to be perturbed by lowering the pH of the medium. Carboxypeptidase Y was rapidly, and essentially irreversibly, inactivated at low p 2H. The pH profile of k cat for the carboxypeptidase Y-catalysed hydrolysis of 4-nitrophenyltrimethylacetate showed two inflections at 45°C: one at p K app, ∼3.7 insensitive to temperature variation (ascribed to a carboxyl group), and one of p K app ∼5.7 markedly temperature-dependent and possibly caused by a histidine residue.