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.
Wragg
S
S
Department of Dental Research, School of Medicine and Dentistry, University of Rochester, New York 14642, USA.
Hagen
F K
FK
Tabak
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C063044:UDP-N-acetylgalactosamine-N-acetylgalactosaminylphosphotransferase