Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.
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.
Tian
Yang
Y
Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
Suk
Dae-Hwan
DH
Cai
Feng
F
Crich
David
D
Mesecar
Andrew D
AD
D000255:Adenosine Triphosphate / Q000378:metabolism; D001408:Bacillus anthracis / Q000201:enzymology*; D040761:Biomimetic Materials / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D003065:Coenzyme A / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D010636:Phenols / Q000378:metabolism; D010654:Phenylbutyrates / Q000378:metabolism; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010636:Phenols; D010654:Phenylbutyrates; C013691:o-succinylbenzoic acid; C109267:4-trifluoromethylphenol; D000255:Adenosine Triphosphate; D013389:Succinate-CoA Ligases; C022693:O-succinylbenzoate - CoA ligase; D003065:Coenzyme A