CALORIMETRIC STUDIES ON CONFORMATIONAL CHANGES IN FLAVOENZYME

对羟基苯甲酸羟化酶(PHBH)是一种含有FAD的酶 催化芳香族化合物的羟基化反应。为了 有效地激活酚类底物进行羟基化 黄素过氧化氢，该酶可使酚类pKA降低约2% 单位，是(至少部分)配体相互作用的结果 酚氧与酪氨酸氢键网络。ITC将成为 用于研究配体与几种PHBH结合时的去质子化 表格，包括WT。滴定将在一定的pH范围内进行 值，得到热力学参数与pH的依赖关系 有约束力的。在任何特定的酸碱度下，滴定将使用 具有不同去质子化热焓的缓冲液。因此，如果 去质子化是配体中的重要组成部分 结合，结合将耦合到缓冲质子化(或 去质子化)，不同的热力学参数 在相同的pH下用不同的缓冲液获得。一个重要的控制， 区分配基去质子化和一种 目前未被怀疑的蛋白质去质子化与配体结合有关， 将是缺少可电离基团的配体的滴定，例如 对氨基苯甲酸酯或苯甲酸酯。存在几个改变pKA的突变体 结合的pOHB(Tyr201Phe，Tyr385 Phe，Asn300Asp，Arg220Lys，Lys297Met， His72Asn)，并将对这些进行研究，以便进一步 了解PHBH如何影响 去质子化pOHB。

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-containing enzyme that catalyzes the hydroxylation of aromatic compounds. In order to efficiently activate the phenolic substrate for hydroxylation by the flavin hydroperoxide, the enzyme lowers the phenolic pKa by about 2 units, a result (at least in part) of the interaction of the ligand phenolic oxygen with a tyrosine hydrogen bonding network. ITC will be used to investigate ligand deprotonation upon binding to several PHBH forms, including WT. Titrations will be conducted over a range of pH values, yielding the pH dependence of the thermodynamic parameters of binding. At any particular pH, titrations will be conducted using buffers which have different enthalpies for deprotonation. Thus, if deprotonations (or protonations) are important components in ligand binding, binding will be coupled to buffer protonation (or deprotonation), and different thermodynamic parameters will be obtained with different buffers at the same pH. An important control, differentiating between ligand deprotonation and the possibility of a currently unsuspected protein deprotonation linked to ligand binding, will be titrations with ligands lacking an ionizable group, such as p-aminobenzoate or benzoate. Several mutants exist that alter the pKa of bound pOHB (Tyr201Phe, Tyr385Phe, Asn300Asp, Arg220Lys, Lys297Met, His72Asn), and these will be studied in order to further our understanding of how PHBH effects the deprotonation of pOHB.