title: Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase from Dictyostelium.
abstract: Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase (Dd-pp alphaGlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-alpha-galactosaminyltransferases, such as murine Mm-pp alphaGalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp alphaGlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp alphaGlcNAcT2 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 alphaGalNAcT1. Two selective competitive inhibitors of Mm-pp alphaGalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp alphaGlcNAcT1 competitively with only slightly less efficacy. Inhibition is specific for Dd-pp alphaGlcNAcT2 relative to two other Dictyostelium retaining glycosyltransferases. These data support a phylogenetic model in which the alphaGlcNAcT function in unicellular eukaryotes converted to an alphaGalNAcT function in the metazoan ortholog while conserving a similar reaction mechanism and active site architecture.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 937, Oklahoma City, OK 73104, USA.
authors.forename: Altan
authors.initials: A
authors.lastname: Ercan
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
mesh_terms: D000818:Animals; D004023:Dictyostelium / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017350:N-Acetylgalactosaminyltransferases