STEADY-STATE KINETICS OF MALONYL-COA SYNTHETASE FROM BRADYRHIZOBIUM-JAPONICUM AND EVIDENCE FOR MALONYL-AMP FORMATION IN THE REACTION
STEADY-STATE KINETICS OF MALONYL-COA SYNTHETASE FROM BRADYRHIZOBIUM-JAPONICUM AND EVIDENCE FOR MALONYL-AMP FORMATION IN THE REACTION
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DOI：
10.1042/bj2970327
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发表时间：
1994-01-15
影响因子：
4.1
通讯作者：
KANG, SW
中科院分区：
文献类型：
--
作者：
KIM, YS;KANG, SW
丙二酰辅酶A合成酶催化丙二酸辅酶A直接从丙二酸和辅酶A生成丙二酰辅酶A，再将三磷酸腺苷水解成AMP和PPI。用稳态动力学方法研究了慢生型根瘤菌丙二酰辅酶A合成酶的催化机理。AMP和PPI的初速度研究和产物抑制研究有力地表明，有序BiUni Uni BiPing Pong Ter Ter体系是丙二酰辅酶A合成酶最可能的稳态动力学机制。三磷酸腺苷、丙二酸和辅酶A的米氏常数分别为61、260和42µM，V-max。为11.2亩/分。T.L.C.在没有辅酶A的情况下，对含有[γ-P-32]ATP的反应混合物中的P-32标记产物的分析表明，PPI是在ATP和丙二酸顺序加成后产生的。通过对P-31-n.m.r的分析，证实了丙二酰AMP的形成是动力学推导机理中的中间产物。从[O-18]丙二酸酯的O-18转移实验中分离出的AMP产物的光谱。P-31-n.m.rAMP产品的信号出现在下午0.024分。除了[O-16(4)]AMP外，表明O-18的一个原子通过丙二酰AMP的形成从[O-18]丙二酸酯转移到AMP上。T.L.C.也证实了丙二酰AMP的形成。含[α-P-32]三磷酸腺苷反应混合物的分析。这些结果有力地支持了从初始速度和产物抑制研究中推导出的有序的Pi Uni Pi Ping Pong Ter Ter机理。
Malonyl-CoA synthetase catalyses,the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PPi. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 mu M, 260 mu M and 42 mu M for ATP, malonate and CoA respectively, and the value for V-max. was 11.2 mu M/min. The t.l.c. analysis of the P-32-labelled products in a reaction mixture containing [gamma-P-32]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of P-31-n.m.r. spectra of an AMP product isolated from the O-18-transfer experiment using [O-18]malonate. The P-31-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [O-16(4)]AMP, indicating that one atom of O-18 transferred from [O-18]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-P-32]ATP. These results strongly support the ordered Pi Uni Uni Pi Ping Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.