Substrate & Inhibitor Binding in Leucine Aminopeptidase

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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.