Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae. Kinetic demonstration of effects of carbohydrate residues on the catalytic mechanism of a glycoenzyme.
In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002268:Carboxypeptidases / Q000378:metabolism*; D002942:Circular Dichroism; D006023:Glycoproteins; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D007527:Isoenzymes; D002268:Carboxypeptidases