Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC

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

Kinetic parameters of the N-acetyltransferase-His8Flag.

1<p>Concentrations were varied between 0.01 and 0.5 mM while the AcCoA concentration was fixed at 2 mM.</p>2<p>Concentrations were varied between 0.083 and 2 mM while the MU-GlcNH₂ concentration was fixed at 1 mM.</p

Immobilized [3H]acetylated N-acetyltransferase-His8Flag intermediate cannot be detected in immunoprecipitation experiments with anti-Flag M2 beads.

1<p>Anti-Flag beads were incubated with extracts from HeLa cell expressing (Transfected) or not expressing (Untransfected) N-acetyltransferase-His8Flag, washed and then incubated with [3H]acetyl-CoA for the given time and pH, at room temperature and with or without 1.3 mM of lipids containing 20% PI.</p>2<p>N-acetyltransferase activity in nmoles of MU produced/hour, and the concentration of protein (pmoles), calculated based on the specific activity of the purified transferase, are given.</p>3<p>The data represent three independent beads binding experiments and assays.</p>4<p>Not detectable.</p

Proteins extracted from HeLa cells permanently expressing N-acetyltransferase-His8Flag were separated by molecular size exclusion chromatography on a 90×1.5 cm Sephacryl S-400 column eluted at 4°C.

<p>Each 1 mL fraction (X-axis) was analyzed for transferase activity (nmol/mL* h, left Y-axis, solid line) and total protein (µg/mL, right Y-axis, dashed line). Additionally every fifth fraction was analyzed for N-acetyltransferase protein by dot-blot using the N-terminal antibody (shown below the X-axis).</p

Direct Michaelis-Menten best-fit curves of specific activity (nmol/h*ng) versus [S] for the N-acetyltransferase reaction with graphic insets displaying the corresponding Lineweaver-Burk double reciprocal plots.

<p>Anti-flag affinity column purified N-acetyltransferase was used and each datum point represents the average of two or three assays. (A) The concentration of MU-GlcNH₂ was varied between 0.01 and 1 mM, while AcCoA was fixed at 0.17 mM (▴), 0.33 mM(○) or 1.0 mM (▪). (B) The concentration of AcCoA was varied between 0.083 and 2.0 mM while MU-GlcNH₂ was fixed at 0.05 mM (⧫), 0.10 mM (□) or 0.30 mM (•). The experiment was independently repeated three times with a representative set of graphics shown.</p