Kim K L, Kang D S, Vitello L B, Erman J E
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Biochemistry. 1990 Oct 2;29(39):9150-9. doi: 10.1021/bi00491a008.
The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.
在pH 7.0和pH 7.5条件下，研究了细胞色素c过氧化物酶催化过氧化氢氧化马心亚铁细胞色素c的稳态动力学，考察了其作为离子强度函数的情况。在固定细胞色素c浓度时，初始速度对过氧化氢浓度的曲线呈双曲线。在低过氧化氢浓度下的极限斜率给出了细胞色素c过氧化物酶 - 过氧化氢反应的表观双分子速率常数，与通过停流技术直接测定的值相同。在过氧化氢饱和（200 μM）时，初始速度对细胞色素c浓度的曲线不是双曲线。速率表达式需要细胞色素c浓度的平方项。该酶的最大周转速率与离子强度无关，在pH 7.0和7.5时的值分别为470±50 s⁻¹和290±30 s⁻¹。速度对细胞色素c浓度曲线的极限斜率为细胞色素c与细胞色素c过氧化物酶氧化中间体之间的缔合速率常数提供了下限。极限斜率从300 mM离子强度下的10⁶ M⁻¹ s⁻¹变化到20 mM离子强度下的10⁸ M⁻¹ s⁻¹，并外推到零离子强度下的5×10⁸ M⁻¹ s⁻¹。根据双结合位点机制和单结合位点多途径机制对数据进行了讨论。
