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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.

SUBMITTER: Noy T

PROVIDER: S-EPMC4066452 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Acetylation of acetyl-CoA synthetase from Mycobacterium tuberculosis leads to specific inactivation of the adenylation reaction.

Noy Tahel T Xu Hua H Blanchard John S JS

Archives of biochemistry and biophysics 20140418

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 character ...[more]

PMID: 24751484

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Project description:Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25 phosphorylation sites on ACSL1. Several unique acetylation and phosphorylation sites occurred under conditions in which fatty acid β-oxidation is normally enhanced. Thirteen of the acetylated lysines had not previously been identified, and none of the phosphorylation sites had been previously identified. Site-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity. In contrast, mutations of Lys285 that mimicked acetylation (Lys285Ala and Lys285Gln) reduced ACSL activity, whereas full activity was retained by Lys285Arg, suggesting that acetylation of Lys285 would be likely to decrease ACSL1 activity. These results indicate that ACSL1 is highly modified post-translationally. Several of these modifications would be expected to alter enzymatic function, but others may affect protein stability or protein-protein interactions.

Dataset Information

Acetylation of acetyl-CoA synthetase from Mycobacterium tuberculosis leads to specific inactivation of the adenylation reaction.

Publications

Acetylation of acetyl-CoA synthetase from Mycobacterium tuberculosis leads to specific inactivation of the adenylation reaction.

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