Substrate & Inhibitor Binding in Leucine Aminopeptidase

基质

• 批准号: 7172309
• 负责人: BRIAN BENNETT
• 金额: $ 17.78万
• 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 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7172309
• 关键词: Active Sites; Amino Acids; Aminopeptidase; Bacteria; Bacterial Infections; Binding; Binding Sites; Catalysis; Catalytic Domain; Classification; Complex; Computer Simulation; Dissociation; Electron Spin Resonance Spectroscopy; Engineering; Enzymes; HIV; Homologous Gene; Human Pathology; Ions; Kinetics; Leucine Aminopeptidase; Localized; Malignant Neoplasms; Maps; Metalloproteins; Metals; Methods; Molecular Target; N-terminal; Numbers; Object Attachment; Peptides; Pharmaceutical Preparations; Preparation; Research Personnel; Sampling; Screening procedure; Site; Site-Directed Mutagenesis; Specificity; Spin Labels; Substrate Interaction; Techniques; Testing; Variant; Vibrio; Vibrio proteolyticus aminopeptidase; analog; antiangiogenesis therapy; chemotherapeutic agent; design; enzyme substrate; inhibitor/antagonist; innovation; microorganism; mutant; programs; tumor

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. cho/erae. 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 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.

拟议的研究计划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的进展 癌症、艾滋病毒和致病性细菌感染。分泌的亮氨酸氨肽酶（VpAP） 蛋白溶弧菌与致病弧菌科的多种氨基肽酶具有高度同源性， 气单胞菌，包括V. cho/cho.氨肽酶与这些细菌的感染性有关。一 与VpAP中的活性位点相邻的疏水性口袋与VpAP中的这种口袋具有结构同源性。 哺乳动物的功能同源物，并可能是底物识别的网站。哺乳动物的酶 是抗肿瘤、免疫调节和抗HIV感染药物的分子靶点。具体 本文提出的目的是为了检验以下假设： 来自溶蛋白弧菌的氨肽酶被邻近的底物结合斑识别， 含催化金属的中心。这些目标与底物结合位点工程相关， 与人类病理学相关抑制剂设计。 具体目标1：证明底物和底物类似物与疏水贴片的结合。 原型氨肽酶（VpAP），来自蛋白弧菌。自旋标记的VpAP和 自旋标记的底物类似物将用于定位VpAP中的底物结合。 具体目标2 "确定VpAP抑制剂和药物的结合常数和动力学参数。 类似底物的动力学参数。抑制剂的Kd和K_的不同值将通过以下获得： EPR光谱和稳态动力学。 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。将获得结构信息 从分析偶极耦合自旋标签和顺磁过渡离子的活性位点。 具体目标4：确定对底物重要的特定酶-底物残基相互作用（即Sn-Pn） 结合性、特异性和方向性。将进行疏水位点突变体的系统动力学研究。

项目成果

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

拟南芥 Glx2-2 催化活性对金属离子的需求。

• DOI: 10.1007/s00775-009-0593-6
• 发表时间: 2010
• 期刊: Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
• 作者: Limphong,Pattraranee;McKinney,RossM;Adams,NicoleE;Makaroff,ChristopherA;Bennett,Brian;Crowder,MichaelW
• 通讯作者: Crowder,MichaelW

Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.

• DOI: 10.1016/j.pep.2009.02.011
• 发表时间: 2009-07
• 期刊: PROTEIN EXPRESSION AND PURIFICATION
• 影响因子: 1.6
• 作者: Hartley, Mariam;Bennett, Brian
• 通讯作者: Bennett, Brian