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 contrar...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3177862?pdf=render |
| _version_ | 1817989369917603840 |
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| author | Xiaolian Fan Ilona Tkachyova Ankit Sinha Brigitte Rigat Don Mahuran |
| author_facet | Xiaolian Fan Ilona Tkachyova Ankit Sinha Brigitte Rigat Don Mahuran |
| author_sort | Xiaolian Fan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T00:46:06Z |
| format | Article |
| id | doaj.art-6330484e8ffc47cd878e57fd6079ff9c |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T00:46:06Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-6330484e8ffc47cd878e57fd6079ff9c2022-12-22T02:22:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0169e2495110.1371/journal.pone.0024951Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC.Xiaolian FanIlona TkachyovaAnkit SinhaBrigitte RigatDon MahuranHeparin 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.http://europepmc.org/articles/PMC3177862?pdf=render |
| spellingShingle | Xiaolian Fan Ilona Tkachyova Ankit Sinha Brigitte Rigat Don Mahuran Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. PLoS ONE |
| title | Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. |
| title_full | Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. |
| title_fullStr | Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. |
| title_full_unstemmed | Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. |
| title_short | Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC. |
| title_sort | characterization of the biosynthesis processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis iiic |
| url | http://europepmc.org/articles/PMC3177862?pdf=render |
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