Substrate & Inhibitor Binding in Leucine Aminopeptidase

超出所提供的空间。拟议的研究计划的长期目标是提供信息，促进有效的，分子靶向特异性化疗药物的设计进展，对癌症，HIM和致病性细菌感染。溶蛋白弧菌分泌的亮氨酸氨肽酶（VpAP）与霍乱弧菌等致病性弧菌科和气单胞菌的多种氨肽酶具有高度同源性。氨肽酶与这些细菌的感染性有关。与VpAP活性位点相邻的疏水口袋与哺乳动物功能同源物中的这种口袋具有结构同源性，并且可能是底物识别的位点。哺乳动物酶是抗肿瘤、免疫调节和抗HIV感染药物的分子靶点。本文提出的具体目的是为了检验这一假设，即来自溶蛋白弧菌的氨肽酶的优选底物被邻近催化含金属中心的底物结合补丁识别。这些目标与人类病理学相关的底物结合位点工程和抑制剂设计有关。具体目标1：证明底物和底物类似物与来自Vibfio Proteolyticus的原型氨肽酶（VpAP）的疏水斑块结合。自旋标记VpAP和自旋标记底物类似物的EPR光谱将用于定位VpAP中的底物结合。具体目标2：确定VpAP抑制剂的结合常数和动力学参数以及类似底物的动力学参数。用EPR谱和稳态动力学方法分别得到了缓蚀剂的Kd值和K_（1/2）。具体目标3：通过VpAP与结合在疏水口袋处的底物和底物类似物的复合物的EPR光谱获得局部结构信息。结构信息将从自旋标记物与活性位点中的顺磁性过渡离子之间的偶极耦合分析中获得。具体目标4：确定对底物结合、特异性和方向重要的特定酶-底物残基相互作用（即Sn-Pn）。将进行疏水位点突变体的系统动力学研究。性能现场=

EXCEED THE SPACE PROVIDED. 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, HIM- and pathogenic bacterial infection. The secreted leucine aminopeptidase (VpAP) from Vibrio proteolyticus has high homology with a number of aminopeptidases from pathogenic vibrionaceae 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, immunomodulatory 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 Vibfio proteolyticus. 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 K_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. PERFORMANCE SITE ========================================Section End===========================================