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Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC.
- Source :
-
PloS one [PLoS One] 2011; Vol. 6 (9), pp. e24951. Date of Electronic Publication: 2011 Sep 21. - Publication Year :
- 2011
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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.
- Subjects :
- Acetyltransferases deficiency
Acetyltransferases isolation & purification
Blotting, Western
Detergents chemistry
Endoplasmic Reticulum metabolism
Enzyme Assays
HeLa Cells
Humans
Hydrophobic and Hydrophilic Interactions
Kinetics
Protein Transport
Temperature
Acetyltransferases biosynthesis
Acetyltransferases metabolism
Mucopolysaccharidosis III enzymology
Protein Processing, Post-Translational
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 6
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 21957468
- Full Text :
- https://doi.org/10.1371/journal.pone.0024951