Backes W L, Eyer C S
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, New Orleans 70112.
J Biol Chem. 1989 Apr 15;264(11):6252-9.
The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.
使用一种重构体系来研究底物对LM2还原的影响，该体系包含二月桂酰磷脂酰胆碱、NADPH-细胞色素P-450还原酶和细胞色素P-450 LM2，其摩尔比为160:1.5:1。一般来说，大多数底物会增加还原第一阶段和第二阶段的速率常数，以及第一阶段中被还原的LM2的比例。如果自旋状态控制着LM2的还原，那么细胞色素的高自旋含量与这些动力学参数之间的相关性会比预期的弱。此外，已表明底物对高自旋含量和还原刺激均有快速影响，这表明低自旋到高自旋的转变并非该特定同工型还原慢相的原因。还在不含LM2和还原酶预形成复合物的含磷脂体系和可溶性体系中研究了细胞色素P-450的还原。在这些体系中，反应比标准重构体系慢得多。添加底物会提高还原速率，这表明添加底物会增加LM2与还原酶之间的结合速率。预形成复合物中LM2的还原速率与LM2和还原酶结合速率的对数之间的强相关性表明，功能性复合物形成的速率是控制还原慢相的因素。
