Kashem M A, Dunford H B
Biochem Cell Biol. 1986 Apr;64(4):323-7. doi: 10.1139/o86-045.
The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.
在25.0摄氏度、离子强度为0.11的水溶液中，使用停流装置和传统分光光度计，研究了辣根过氧化物酶化合物I和II（HRP-I和HRP-II）氧化还原型烟酰胺腺嘌呤二核苷酸（NADH）的瞬态动力学，作为pH的函数。与使用许多其他底物的研究一致，HRP-I-NADH反应的pH依赖性可以用HRP-I活性位点pKa = 4.7 +/- 0.5的单一电离来解释。与其他底物的研究相反，HRP-II-NADH反应的pH依赖性可以用HRP-II活性位点pKa为4.2 +/- 1.4的单一电离来解释。观察到HRP-II-NADH反应具有明显的可逆性。在4-10的pH范围内，HRP-I与NADH反应的速率常数从2.6×10⁵到5.6×10² M⁻¹ s⁻¹不等，HRP-II与NADH反应的速率常数从4.4×10⁴到4.1 M⁻¹ s⁻¹不等。为了定量解释辣根过氧化物酶与NADH的氧化酶反应，必须考虑这些速率常数。
