Ogusucu Renata, Rettori Daniel, Munhoz Daniela Cristina, Netto Luis Eduardo Soares, Augusto Ohara
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Free Radic Biol Med. 2007 Feb 1;42(3):326-34. doi: 10.1016/j.freeradbiomed.2006.10.042. Epub 2006 Oct 20.
Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.
过氧化物酶作为抗氧化损伤的防御者和过氧化氢介导的信号事件的传感器，正受到越来越多的关注。对这两种功能可能至关重要的是与过氧化氢的快速反应，其二级速率常数通常高于10⁵ M⁻¹ s⁻¹。然而，直到最近，这些速率常数报道的值仍在10⁴ - 10⁵ M⁻¹ s⁻¹范围内，包括来自酿酒酵母的胞质硫氧还蛋白过氧化物酶I（Tsa1）和II（Tsa2）的速率常数。为了解决这一明显的矛盾，我们开发了一种与辣根过氧化物酶竞争的动力学方法，以确定过氧化物酶与过氧亚硝酸盐和过氧化氢反应的二级速率常数。该方法经过验证，可用于测定在pH 7.4、25℃条件下Tsa1和Tsa2与过氧亚硝酸盐（k约为10⁵ M⁻¹ s⁻¹）和过氧化氢（k约为10⁷ M⁻¹ s⁻¹）反应的二级速率常数。它还能分别测定Tsa1和Tsa2的过氧化物半胱氨酸（Cys47）的pKa为5.4和6.3。除了提供一种研究硫醇蛋白动力学的有用方法外，我们的研究还补充了最近的报道，这些报道对流行观点提出了挑战，即与血红素和硒蛋白相比，过氧化物酶对过氧化氢的催化能力较差。
