摘要
己知动物脂肪酸合成酶的底物乙酰辅酶A和丙二酰辅酶A具有竞争性双底物抑制的乒乓机制。实验发现鸭肝脂肪酸合成酶的第三个底物NADPH也具有底物抑制,并研究了它的规律及与NADPH有关的稳态动力学。发现对于该酶的全反应,增加丙二酰辅酶A浓度,降低环境盐浓度,均使NADPH底物抑制减少。但以NADPH作底物的酮酰还原和烯酰还原二步单独反应以及包含四步单独反应的乙酰乙酰辅酶A还原反应都无NADPH底物抑制现象。NADPH底物抑制对丙二酰辅酶A为竞争性,丙二酰辅酶A底物抑制对NADPH为非竞争性。在全反应中NADPH和丙二酰辅酶A之间发现为乒乓机制,在乙酰乙酰辅酶A还原反应中,两个底物NADPH和乙酰乙酰辅酶A之间则表现为序列反应机制。降低环境盐浓度使NADPH和丙二酰辅酶A之间的乒乓机制向序列机制转化。在全反应中,NADP产物抑制相对NADP为竞争性,对丙二酰辅酶A为非竞争性。
Overall activity of duck liver fatty acid synthase is inhibited by high concentrations of NADPH in the case of low concentration of malonyl CoA. This substrate inhibition decreases in high concentration of malonyl CoA or low PH. In the same conditions no inhibition by NADPH is detected in the reaction of ketoreduction, enoyl reduction and acetoacetyl CoA reduction. The inhibition by NADPH is competitive with malonyl CoA, but the inhibition by malonyl CoA is non-competitive with NADPH. The studies for steady state kinetics show that malonyl CoA and NADPH follow a ping-pong mechanism, however acetyl CoA and NADPH do not. Low salt concentrations or low PH causes the ping-pong mechanism between malonly CoA and NADPH transfer to sequential mechanism. The product inhibitionby NADP is competitive with NADPH and is non-competitive with malonyl CoA. It is suggested that the binding of NADPH to FAS is not random, which must occur after condensation. The NADPH substrate inhibition is due to the earlier binding of NADPH to the enzyme. It is assumed that the earlier NADPH binding to FAS causes the rotating arm move from condensation domain to reduction domain so as to affect the condensation reaction and inhibit the activity. More positive charges in the condensation center, caused by low salt concentration or low PH, will strengthen its interaction with the negatively charged malonyl group carried on the rotating arm , therefore may favour the condensation reaction and resist NADPH inhibition.
基金
国家教委优秀年轻教师基金
关键词
酶动力学
脂肪酸合成酶
鸭
NADPH
Enzyme kinetics
Substrate inhibition
Fatty acid synthase
Product inhibiton