Substrate & Inhibitor Binding in Leucine Aminopeptidase

描述（由申请人提供）：拟议研究项目的长期目标是提供信息，促进设计有效的、分子靶向特异性化疗药物，治疗癌症、HIV和致病性细菌感染。proteo/yticus弧菌分泌的亮氨酸氨基肽酶（VpAP）与致病性弧菌科和气单胞菌（包括霍乱弧菌）的许多氨基肽酶具有高度同源性。氨基肽酶与这些细菌的传染性有关。VpAP活性位点附近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，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感染药物的分子靶点。本文提出的具体目的是为了验证一种假设，即来自溶蛋白弧菌的氨基肽酶的首选底物被靠近催化含金属中心的底物结合斑块识别。这些目标与底物结合位点工程和与人类病理相关的抑制剂设计有关。具体目标1：证明底物和底物模拟物与来自蛋白质弧菌/细胞弧菌的原型氨基肽酶（VpAP）的疏水性斑块结合。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中底物的结合。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。抑制剂的Kd和Ki值将分别通过EPR光谱和稳态动力学得到。

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.