Regulation of Xanthine Oxidase Activity by Substrates at Active Sites via Cooperative Interactions between Catalytic Subunits: Implication to Drug Pharmacokinetics
作者:L. A. Tai、K. C. Hwang
DOI:10.2174/092986711793979760
日期:2011.1.1
Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a “substrate inhibition” pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a “substrate activation” pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s-1 to 10.5 s-1, and converts the V-[S] plot from “substrate inhibition” pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.
三种黄嘌呤氧化酶底物(即黄嘌呤、腺嘌呤和2-氨基-4-羟基吡啶)表现出“底物抑制”模式(即在较高底物浓度下,转化速率较慢),而另两种底物(即黄吡啶和光亮素)则表现出“底物激活”模式(即在较高底物浓度下,转化速率较高)。在两个黄嘌呤氧化酶活性位点中的一个结合6-形式吡啶会使邻近活性位点的黄嘌呤转化速率从17.0 s-1减慢至10.5 s-1,并将V-[S]图从“底物抑制”模式转换为经典的米氏-孟德尔曲线饱和模式。相反,在活性位点结合黄嘌呤会加速邻近活性位点6-形式吡啶的转化速率。实验结果表明,底物可以通过在活性位点的结合调节黄嘌呤氧化酶的活性;同时,黄嘌呤氧化酶的催化亚单位也可以作为调节单位。基于从扩展米氏-孟德尔模型推导出的速率方程的理论模拟表明,V-[S]图中的底物抑制和底物激活行为可以通过引入同源二聚体酶中两个催化亚单位之间的协同作用来获得。目前的研究确认了黄嘌呤氧化酶的两个催化亚单位之间存在非常强的协同作用。