title: Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC.
abstract: Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction.
authors.affiliation: Genetics and Genome Biology Program, The Hospital For Sick Children, Toronto, Canada.
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Fan
authors.forename: Ilona
authors.initials: I
authors.lastname: Tkachyova
authors.forename: Ankit
authors.initials: A
authors.lastname: Sinha
authors.forename: Brigitte
authors.initials: B
authors.lastname: Rigat
authors.forename: Don
authors.initials: D
authors.lastname: Mahuran
mesh_terms: D000123:Acetyltransferases / Q000096:biosynthesis* / Q000172:deficiency / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D003902:Detergents / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057075:Enzyme Assays; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009084:Mucopolysaccharidosis III / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D013696:Temperature
chemical_list: D003902:Detergents; D000123:Acetyltransferases; C514442:HGSNAT protein, human