Substrate & Inhibitor Binding in Leucine Aminopeptidase

基质

• 批准号: 6677111
• 负责人: BRIAN BENNETT
• 金额: $ 18.75万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677111
• 关键词: Vibrio; active sites; aminopeptidase; antiAIDS agent; antibacterial agents; antineoplastics; chemical kinetics; drug design /synthesis /production; electron spin resonance spectroscopy; enzyme inhibitors; enzyme substrate analog; enzyme substrate complex; gene mutation; hydropathy; organometallic compounds; protein binding; protein structure function

DESCRIPTION (provided by applicant): The long term objective of the proposed program of study is to provide information that facilitates advances in the design of potent, molecular target-specific chemotherapeutic agents against cancers, HIV- and pathogenic bacterial infection. The secreted leucine aminopeptidase (VpAP) from Vibrio proteo/yticus has high homology with a number of aminopeptidases from pathogenic vibdonaceae and aeromonads, including V. cholerae. The aminopeptidase is implicated in infectivity of these bacteria. A hydrophobic pocket adjacent to the active site in VpAP has structural homology with such a pocket in mammalian functional homologues and may be the site of substrate recognition. The mammalian enzymes are the molecular targets for anti-tumor, immunornodulatory and anti-HIV infectivity drugs. The specific aims presented herein are designed to test the hypothesis that preferred substrates of the aminopeptidase from Vibrio proteolyticus are recognized by a substrate-binding patch adjacent to the catalytic metal-containing center. These aims are pertinent to substrate-binding-site engineering and inhibitor design relevant to human pathologies. Specific Aim 1: Demonstrate substrate and substrate analog binding to the hydrophobic patch of the prototypical aminopeptidase (VpAP) from Vibrio proteo/yticus. EPR spectroscopy of spin labeled VpAP and spin labeled substrate analogs will be used to localize substrate binding in VpAP. Specific Aim 2: Determine the binding constants and kinetic parameters for inhibitors of VpAP and the kinetic parameters of analogous substrates. Distinct values for Kd and Ki for inhibitors will be obtained by EPR spectroscopy and steady state kinetics, respectively. Specific Aim 3: Obtain local structural information through EPR spectroscopy of complexes of VpAP with substrates and substrate analogs bound at the hydrophobic pocket. Structural information will be obtained from analysis of dipolar couplings between spin labels and paramagnetic transition ions in the active site. Specific Aim 4: Identify specific enzyme-substrate residue interactions (i.e. Sn-Pn) important for substrate binding, specificity and orientation. Systematic kinetic studies of hydrophobic site mutants will be carried out.

描述（由申请人提供）：拟议的研究计划的长期目标是提供信息，以促进针对癌症，HIV和致病细菌感染的有效的，分子靶标特异性化学治疗剂的设计进展。 来自Vibrio Proteo/Yticus的分泌的亮氨酸氨基肽酶（VPAP）具有较高的同源性，其中许多来自致病性乙酰曲科和气管的氨基肽酶（包括V. cholerae）。 氨基肽酶与这些细菌的感染性有关。 VPAP中活性位点附近的疏水口袋具有结构同源性，具有这样的哺乳动物功能同源物中的口袋，并且可能是底物识别的位置。 哺乳动物酶是抗肿瘤，免疫调节和抗HIV感染性药物的分子靶标。 本文介绍的特定目的旨在测试假设，即从弧菌蛋白水解的氨基肽酶的首选底物通过与含催化金属的中心相邻的底物结合贴片识别。 这些目标与与人体病理有关的底物结合位点工程和抑制剂设计有关。具体目标1：证明来自Vibrio proteo/Yticus的底物和底物类似物结合与原型氨基肽酶（VPAP）的疏水片结合。 自旋标记为VPAP和自旋标记的底物类似物的EPR光谱将用于将底物结合在VPAP中进行定位。 具体目标2：确定VPAP抑制剂和类似底物的动力学参数的结合常数和动力学参数。 EPR光谱和稳态动力学将获得KD和KI的不同值。 特定目标3：通过VPAP复合物与底物和底物类似物结合在疏水袋中的底物的EPR光谱获得局部结构信息。 结构信息将从对活性位点中自旋标记和顺磁过渡离子之间的偶极耦合分析获得。 特定目标4：确定特定的酶 - 基底残基相互作用（即SN-PN）对于底物结合，特异性和方向很重要。 将进行疏水性位点突变体的系统动力学研究。