Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alphaglucosaminyltransferase from Dictyostelium. Glycobiology

ow nloaded from 2Abstract Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-α-glucosaminyltransferase (Dd-pp αGlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-α-galactosaminyltransferases such as murine Mm-pp αGalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp αGlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp αGlcNAcT2 strongly prefers UDP-GlcNAc over UDP-GalNAc, preferentially modifies the central region of the peptide, and modifies Ser- in addition to Thr-residues. Initial velocity measurements performed over a matrix of UDP-GlcNAc donor and peptide acceptor concentrations indicate that the substrates bind to the enzyme in ordered fashion before the chemical conversion. Substrate inhibition exerted by a second peptide, and the pattern of product inhibition exerted by UDP, suggest that UDP-GlcNAc binds first and the peptide binds second, consistent with data reported for Mm-pp αGalNAcT1. Two selective competitive inhibitors of Mm-pp αGalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp αGlcNAcT1 competitivel