Mechanism Based Design of Novel Inhibitors of Dinuclear Hydrolases

这一奖项由无机化学、生物无机化学和金属有机化学以及分子生物化学计划颁发，支持犹他州立大学理查德·B·霍尔茨教授的研究，目的是确定蛋白质溶解单胞菌的亮氨酸氨基肽酶和流感嗜血杆菌编码的N-琥珀酰基-L，L二氨基甲酸脱丁二酸酶(DAPE)的详细催化机理，并设计和合成有效的DAPE抑制剂。。这项研究涉及一种跨学科的方法，结合了生化、光谱和X射线结晶学方法以及合成DAPE的小分子抑制剂。本工作涉及五个关键方面：(1)AAP活性中心的哪些残基起着重要的催化作用？(2)发展了Laue X射线衍射法来研究AAP的催化机理。(3)底物和过渡态类似物抑制剂与DAPE相互作用的表征。(4)DAPE活性中心残基修饰酶的制备。(5)含有双核金属中心的AAP和DapE水解酶的详细作用机制的发展是许多生物过程的核心，因此，表征它们的结构和功能是一个非常重要的问题。它们在组织修复、蛋白质成熟、激素水平调节、细胞周期控制以及细胞壁合成等方面发挥着重要作用。一些双核氨基肽酶具有广泛的底物特异性，广泛分布于植物和动物组织中。由于它们在蛋白质和生物活性多肽(包括激素)的降解中发挥作用，它们具有广泛的生物学和药用意义。在肿瘤细胞表面观察到亮氨酸氨基肽酶的活性，这一事实突显了了解亮氨酸氨基肽酶反应机制的重要性，而且这种活性在肿瘤发生中起着关键作用。此外，天然的多肽类似物Bestatin通过抑制亮氨酸氨基肽酶的活性显著降低男性的艾滋病毒感染。对于DAPE来说，对目前所有可用的抗生素都有抗药性的几种病原菌的出现强调了它们的重要性，因为MDAP/赖氨酸生物合成途径的两种产物MDAP和赖氨酸都是革兰氏阴性和大多数革兰氏阳性细菌肽聚糖细胞壁的基本成分。本研究旨在详细了解双核金属中心催化的水解反应，这可能会导致新抗生素的开发，并为本科生、研究生和博士后的发展提供支持。

This award by the Inorganic, Bioinorganic and Organometallic Chemistry and the Molecular Biochemistry programs supports research by Professor Richard B. Holz at Utah State University to determine a detailed catalytic mechanism for the leucine aminopeptidase from Aeromonas proteolytica (AAP) and the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from H. influenzae and to design and syntheize potent inhibitors of DapE. . This research involves an interdisciplinary approach that incorporates biochemical, spectroscopic, and X-ray crystallographic methods and the synthesis of small molecule inhibitors of DapE. The work addresses five key areas: (1) Which residues in the active site of AAP play catalytically important roles? (2) The development of Laue X-ray diffraction methods to study the catalytic mechanism of AAP. (3) Characterization of how substrate- and transition-state analog inhibitors interact with DapE. (4) Preparation of altered enzymes of proposed active site residues in DapE. (5) The development of a detailed mechanism of action for AAP and DapEHydrolases that contain dinuclear metal centers are central to numerous biological processes and, consequently, characterization of their structure and function is a problem of outstanding importance. They play important roles in tissue repair, protein maturation, hormone level regulation, cell-cycle control, as well as cell wall synthesis. Several dinuclear aminopeptidases have broad substrate specificities and are widely distributed in both plant and animal tissues. Their biological and medicinal significance is extensive due to their roles in the degradation of proteins and biologically active peptides, including hormones. The importance of understanding the reaction mechanism of leucine aminopeptidases is underscored by the fact that leucine aminopeptidase activity has been observed on the surface of tumor cells, and such activity is known to play key roles in oncogenesis. In addition, the naturally occurring peptide analog inhibitor, bestatin, significantly decreases HIV infection in males by inhibiting leucine aminopeptidase activity. For DapE's, the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics emphasize their importance, since both products of the mDAP/lysine biosynthetic pathway, mDAP and lysine, are essential components of the peptidoglycan cell wall for Gram-negative and most Gram-positive bacteria. This research aims toward a detailed understanding of hydrolytic reactions catalyzed by dinuclear metal centers, which may lead to the development of new antibiotics and also supports the development of undergraduate, graduate and postdoctoral students.