Rodríguez-López J N, Gilabert M A, Tudela J, Thorneley R N, García-Cánovas F
Grupo de Investigación de Enzimología (GENZ), Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Biochemistry. 2000 Oct 31;39(43):13201-9. doi: 10.1021/bi001150p.
Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.
对2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)与辣根过氧化物酶(HRPC)化合物II反应的双相单周转数据进行瞬态动力学分析，结果表明在限速电子转移(k(+6)=42.1 s(-1))之前，ABTS存在预平衡结合(k(+5)=7.82×10(4) M(-1) s(-1))。这些数据是使用停流法获得的，该方法在反应介质中加入抗坏血酸，以维持ABTS的低稳态浓度(准一级条件)，并使由于ABTS阳离子自由基积累而导致的索雷特区域吸光度变化最小化。对该反应的稳态动力学分析证实，该底物对HRPC化合物II的还原在完整的过氧化物酶循环中是限速步骤。使用计时法研究了HRPC与邻二酚的反应，该方法在测定介质中也加入了抗坏血酸，以最小化涉及酚衍生自由基产物的非酶反应的影响。这使得能够根据抗坏血酸的存在引起的延迟期来确定不同过氧化氢和邻二酚浓度下邻二酚氧化的初始速率。动力学分析将HRPC化合物II与邻二酚的反应分解为两个步骤，首先形成酶-底物复合物，然后电子从底物转移到血红素。对于快速氧化的邻二酚，血红素结合的过氧化氢的O-O键的异裂(k(+2)=2.17×10(3) s(-1))是限速步骤。邻二酚底物的大小和疏水性与其与HRPC的结合速率相关，而C-4羟基处的电子密度主要影响电子转移到血红素的速率。
