Pre-steady State Kinetic Analysis of cis-3-Chloroacrylic Acid Dehalogenase

顺-3-氯丙烯酸脱卤酶的预稳态动力学分析

• 批准号: 7932955
• 负责人: Gottfried Karl Schroeder
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932955
• 关键词: Acids; Active Sites; Address; Affect; Affinity; Alanine; Amino Acid Sequence; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Binding; Biological Models; Catalysis; Characteristics; Chemistry; Data; Development; Discrimination; Distal; Drug usage; Elements; Enzymes; Evolution; Exhibits; Felis catus; Goals; Health; Homologous Gene; Human; Hydrogen Bonding; Imidazole; Individual; Isomerism; Kinetics; Knowledge; Lead; Light; Methods; Motion; Mutation; Nature; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Public Health; Reaction; Research; Resistance; Resources; Role; Side; Source; Specificity; Study models; Substrate Specificity; Thermodynamics; Work; base; cis-3-chloroacrylic acid dehalogenase; combat; drug modification; enzyme activity; enzyme mechanism; flexibility; improved; inhibitor/antagonist; insight; member; mutant; novel strategies; public health relevance; response; simulation

DESCRIPTION (provided by applicant): The long term goal of this research is to obtain a better understanding of the origins of enzyme specificity and catalysis and how loop regions and residues distal to the active site contribute to catalysis and substrate selectivity. Our research utilizes two enzymes that are members of the same superfamily, cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and a cis-CaaD homologue designated CgX. These enzymes share 54% sequence similarity and the key elements of the catalytic machinery. Yet, CgX is a poor cis-CaaD as reflected by its low efficiency (processing the cis-isomer) and lack of specificity (processing both isomers). The reasons for these differences are not known, yet this information could shed light on the evolution of isomer specificity in the cis- and frans-3-chloroacrylic acid dehalogenases, lead to a better understanding of the characteristics of the tautomerase superfamily, and advance our knowledge of enzymes. The aims of this proposal will assess the influence of protein motion, loop identity, and distal hydrogen bonding networks involving active site residues on each step of the catalytic cycle. Our two major aims are (1) to determine the effects of the individual replacement of the six amino acids of the flexible cis-CaaD active site loop with those of CgX on both steady-state and pre-steady state kinetic parameters; and (2) to examine the role of the critical cis-CaaD residue His28 in substrate binding by investigating the effects of indirect and direct mutation(s) on the kinetics of both binding and catalysis. Kinetic data will be globally fit to a single mechanism by simulation to resolve individual rate constants. This approach will allow the effects of mutational replacements on binding, chemistry, conformational changes, and/or product release steps to be critically evaluated. These studies, combined with other results, could form the basis for a well-defined model for the study of enzyme specificity and become a valuable resource for developing new approaches to combat antibiotic resistance. PUBLIC HEALTH RELEVANCE: Antibiotic resistant bacteria have become a major public health threat and will continue to be one. It is therefore critical to understand the origins of enzyme specificity and how it changes as a result of mutations; as this is one way bacteria acquire abilities that render them less susceptible to antibiotic treatments.

描述(申请人提供)：这项研究的长期目标是更好地了解酶的专一性和催化的起源，以及活性部位远端的环区和残基如何有助于催化和底物选择性。我们的研究利用了两种属于同一超家族成员的酶，顺-3-氯丙烯酸脱卤酶(cis-CAAD)和一个命名为CGX的顺式-CAAD同源物。这些酶有54%的序列相似性和催化机械的关键元素。然而，CGX是一种很差的顺式CAAD，这反映在它的效率低(处理顺式异构体)和缺乏特异性(处理两种异构体)。这些差异的原因尚不清楚，但这些信息可能有助于阐明顺式和法式氯丙烯酸脱卤酶的异构体专一性的进化，有助于更好地理解互变擦除酶超家族的特征，并促进我们对酶的了解。这项建议的目的是评估蛋白质运动、环特性和涉及活性中心残基的远端氢键网络对催化循环每一步的影响。我们的两个主要目标是(1)确定用CGX的活性部位环中的6个氨基酸单独替换对稳态和稳态前动力学参数的影响；(2)通过研究间接突变和直接突变(S)对结合动力学和催化动力学的影响，研究关键的顺式CAAD残基His28在底物结合中的作用。通过模拟将动力学数据全局地拟合到单个机制，以求出单独的速率常数。这种方法将允许对突变替换对结合、化学、构象变化和/或产物释放步骤的影响进行严格的评估。这些研究与其他结果相结合，可能形成一个明确的酶特异性研究模型的基础，并成为开发对抗抗生素耐药性的新方法的宝贵资源。与公共健康相关：抗药性细菌已经成为并将继续成为一个主要的公共健康威胁。因此，了解酶特异性的起源以及它是如何由于突变而变化的是至关重要的；因为这是细菌获得使其不太容易受到抗生素治疗的能力的一种方式。