Frederick Kendra King, Palfey Bruce A
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2005 Oct 11;44(40):13304-14. doi: 10.1021/bi051119t.
p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.
对羟基苯甲酸羟化酶（PHBH）是一种依赖黄素腺嘌呤二核苷酸（FAD）的单加氧酶，在依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的反应中催化对羟基苯甲酸（pOHB）羟基化为3,4 - 二羟基苯甲酸。有两个结构特征相互关联以控制PHBH与NADPH的反应活性：一个质子转移网络，它允许质子在隔离的活性位点和溶剂之间传递；以及一种黄素，它具有两个位置：“在位”，此时黄素靠近pOHB；“离位”，此时黄素靠近NADPH。PHBH在允许黄素采用可接近NADPH的构象之前，利用质子转移网络来检测合适的芳香底物是否存在。在这项工作中，对His72Asn突变体（其质子转移网络被破坏）的动力学分析表明，无论有无NADPH，黄素都可能发生移动，但NADPH会刺激其移动到氢化物转移所需的反应性构象。底物和溶剂同位素效应作用于His72Asn突变体还原的瞬态动力学，结果表明质子转移与黄素移动相关联，并且构象变化发生在与氢化物转移不同的步骤中。通过在无缓冲溶液中存在荧光pH指示剂染料的情况下进行实验，直接在野生型酶中观察到了还原半反应过程中的质子转移。NADPH结合导致酶快速释放质子，随后在黄素还原后再摄取质子。溶剂和底物动力学同位素效应表明，在野生型PHBH中，质子偶联的黄素移动和还原也发生在不同的步骤中。这些结果使得能够为野生型酶的还原半反应提出一个详细的动力学方案附录中分析了考虑底物诱导异构化的三种动力学模型。
