CALORIMETRIC STUDIES ON CONFORMATIONAL CHANGES IN FLAVOENZYME

黄酶构象变化的量热研究

• 批准号: 6122027
• 负责人: DAVID P BALLOU
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-05 至 1998-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6122027
• 关键词: biological products; biomaterials; biomedical resource; education; human tissue; model design /development; technology /technique development

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.

对羟基苯甲酸羟化酶（PHBH）是一种含FAD的酶 催化芳香族化合物的羟基化。 为了 有效地活化酚类底物， 黄素氢过氧化物，酶降低酚pKa约2 单位，配体相互作用的结果（至少部分） 具有酪氨酸氢键网络的酚氧。 ITC将 用于研究与几种PHBH结合后的配体去质子化 包括WT。 将在一定的pH值范围内进行滴定 值，产生热力学参数的pH依赖性， 约束力 在任何特定pH值下，滴定将使用 缓冲液具有不同的去质子化能力。 因此，如果 去质子化（或质子化）是配体中的重要组成部分， 结合，结合将偶联到缓冲液质子化（或 去质子化），并且不同的热力学参数将被 在相同pH下用不同缓冲液获得。一个重要的对照， 区分配体去质子化和 目前未被怀疑的蛋白质去质子化与配体结合有关， 将是用缺乏可电离基团的配体滴定，例如 对氨基苯甲酸酯或苯甲酸酯。 存在几种突变体， 结合的pOHB（Tyr201Phe，Tyr385Phe，Asn300Asp，Arg220Lys，Lys297Met， His72Asn），这些将被研究，以进一步我们的 了解PHBH如何影响 pOHB的去质子化。