Kinetic analysis of a recombinant UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase.

A mammalian expression vector was designed to express a secreted soluble form of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) with a metal binding site (HHWHHH) at the NH2 terminus. The recombinant enzyme was purified to homogeneity from COS-7 cell media by sequential chromatography on columns of NiCl2-chelating Sepharose, Affi-Gel blue, and Sephacryl S-100. Kinetic parameters of recombinant and native polypeptide GalNAc transferase were comparable for the donor UDP-GalNAc and for the peptide acceptor AcTPPP, EPO-T (PPDAATAAPLR), and HVF (PHMAQVTVGPGL). Initial velocity and product inhibition studies were carried out with purified recombinant polypeptide GalNAc transferase and the substrates UDP-GalNAc and peptide EPO-T. Initial velocity data was consistent with a sequential type mechanism in which binding of both substrates precedes product release. Product inhibition analysis using UDP showed competitive inhibition against UDP-GalNAc and a noncompetitive inhibition against peptide EPO-T. The dead end peptide analogue EPO-G (PPDAAGAAPLR) was a noncompetitive inhibitor of UDP-GalNAc and a competitive inhibitor of peptide EPO-T. Collectively, the results suggest that the most probable kinetic mechanism for the enzyme is one in which both substrates must bind in a random order prior to catalysis. Interestingly, the Km for EPO-T is similar to the Ki for EPO-G, suggesting that peptide interaction with the polypeptide GalNAc transferase does not require a hydroxyamino acid.