title: Acetylation of acetyl-CoA synthetase from Mycobacterium tuberculosis leads to specific inactivation of the adenylation reaction.
abstract: Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A, allowing the organism to grow on acetate as the sole carbon source. ACS was the first enzyme in Mycobacterium tuberculosis shown to be regulated by posttranslational acetylation by the cAMP-dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD(+) and a mycobacterial sirtuin-like deacetylase. In this study we characterize the kinetic mechanism of MtACS, where the overall reaction can be divided into two half-reactions: the acetyl-adenylate forming reaction and the thiol-ligation reaction. We also provide evidence for the existence of the acetyl-adenylate intermediate via (31)P NMR spectroscopy. Furthermore, we dissect the regulatory role of K617 acetylation and show that acetylation inhibits only the first, adenylation half-reaction while leaving the second half reaction unchanged. Finally, we demonstrate that the chemical mechanism of the enzyme relies on a conformational change which is controlled by the protonation state of aspartate 525. Together with our earlier results, this suggests a degree of regulation of enzyme activity that is appropriate for the role of the enzyme in central carbon metabolism.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park avenue, Bronx, NY 10461, United States.
authors.forename: Tahel
authors.initials: T
authors.lastname: Noy
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park avenue, Bronx, NY 10461, United States.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park avenue, Bronx, NY 10461, United States. Electronic address: john.blanchard@einstein.yu.edu.
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000106:Acetate-CoA Ligase / Q000235:genetics / Q000378:metabolism*; D019342:Acetic Acid / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011522:Protons; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D011994:Recombinant Proteins; C017987:acetyl adenylate; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000106:Acetate-CoA Ligase; D019342:Acetic Acid
keywords: Central carbon metabolism; Enzyme kinetics; Enzyme regulation; Tuberculosis
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