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Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

ABSTRACT: The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg(2+) results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg(2+) and ATP are varied together.

PROVIDER: S-EPMC1221953 | BioStudies | 2001-01-01

REPOSITORIES: biostudies

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Project description:UNLABELLED:Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE:Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Project description:o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of enzymes.

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Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

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