Tian Yang, Suk Dae-Hwan, Cai Feng, Crich David, Mesecar Andrew D
Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
Biochemistry. 2008 Nov 25;47(47):12434-47. doi: 10.1021/bi801311d.
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.
邻琥珀酰苯甲酰辅酶A（OSB-CoA）合成酶（EC 6.2.1.26）催化邻琥珀酰苯甲酸（OSB）与辅酶A在ATP依赖下缩合形成OSB-CoA，这是炭疽芽孢杆菌中甲萘醌生物合成途径的第四步。基因敲除研究已将这种酶突显为发现新型抗生素的潜在靶点。在此，我们报告了对炭疽芽孢杆菌OSB-CoA合成酶动力学机制的首次研究，将其归类为有序双单双乒乓机制。通过一系列预稳态和稳态动力学研究并结合直接结合研究，结果表明，作为最后结合的底物，辅酶A在第一轮周转后强烈激活前半反应。前半反应的激活很可能是通过辅酶A稳定酶处于“F”形式的构象来实现的，该构象会缓慢异构化为E形式。因此，OSB-CoA合成酶的动力学机制可能更准确地描述为有序双单双异构乒乓机制。使用一系列羧酸基团有改变的OSB类似物探究了OSB-CoA合成酶的底物特异性。OSB-CoA对OSB的偏好明显高于所有测试的类似物，因为除了4-[2-(三氟甲基)苯基]-4-氧代丁酸外，其他类似物均无活性，而该类似物的k(cat)/K(m)降低了100倍。基于对OSB-CoA合成酶动力学机制和底物特异性的理解，设计并合成了OSB-AMP的反应中间体类似物5'-O-{N-[2-(三氟甲基)苯基]-4-氧代丁基}腺苷磺酰胺（TFMP-丁基-AMS）。发现该抑制剂对辅酶A是反竞争性抑制剂，对ATP和OSB是混合型抑制剂，抑制常数为低微摩尔级。总体而言，这些结果应为更详细、广泛的抑制剂设计研究充当重要的先导，这些研究旨在开发针对OSB-CoA合成酶类酶的先导化合物。
