Dunford H B, Cotton M L
J Biol Chem. 1975 Apr 25;250(8):2920-32.
The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.
在离子强度为0.11的水溶液中，于25℃下，对辣根过氧化物酶化合物I和II催化对氨基苯甲酸氧化的动力学进行了深入研究，该研究以pH为函数。所有速率数据均从涉及单一酶化合物反应的单周转实验中收集。在两种化合物的反应中，在高pH值下均观察到相对于酶的一级行为偏差，这是根据产物与酶的自由基相互作用来解释的。通过足够过量的底物可以消除这种影响。在低pH下观察到的底物一级动力学行为偏差，是由一种涉及酶 - 底物复合物的机制来解释的，该复合物与额外的底物分子反应，但速率较慢。对氨基苯甲酸与游离化合物I和II反应的二级速率常数的pH依赖性，与亚铁氰化物可比反应的结果相似，这表明两种还原底物具有相似的质子转移机制。对氨基苯甲酸对化合物II的还原似乎受到酶上两个可电离基团的影响，这两个基团影响血红素的电子环境。底物可电离基团对化合物II反应速率缺乏影响，表明在pH的酸性区域，NH2C6H4COO-和NH2C6H4COOH反应活性的潜在差异因扩散控制极限而趋于平衡。对氨基苯甲酸对化合物I的还原不受扩散控制，其速率 - pH曲线可以用三个酸电离来解释，其中两个在底物上，一个在化合物I上。
