Role of Binding Determinants in Enzyme Catalysis

酶是加速（催化）生物反应的蛋白质。我们的研究重点是了解酶与其作用的底物之间发生的相互作用如何产生催化作用。这些知识对于了解酶如何工作、设计新的酶催化剂以及设计酶抑制剂（药物/除草剂）至关重要。首先，我们研究扁桃酸消旋酶（MR），其催化D-和L-扁桃酸的相互转化，并作为理解酶如何催化不利反应（即，C-H键裂解）。消旋酶和差向异构酶由于其用于制备D-氨基酸、稀有糖和其他增值化合物而引起极大兴趣。这个建议通过4个主题扩展了我们对酶的研究。主题[1]着重于用NMR活性核标记消旋酶的催化性酸碱残基以区分它们的质子化状态。这些研究将提供直接的证据，底物结合如何改变酸碱催化剂在消旋酶的活性位点的pKa。这些知识有助于合理设计新型酶催化剂。主题[2]阐述了我们开发的两种酶抑制策略：硼酸抑制作用于碳酸底物的酶和底物产物类似物（SPAs）抑制消旋酶。我们将研究热力学和结构方面的抑制MR硼酸，以澄清如何硼酸为基础的抑制剂与这些酶相互作用。结果将是感兴趣的制药，因为硼酸基团提供了一个点的分子识别的药物，并表现出低毒性。我们将合成SPA以获得非核糖体肽合成酶（NRPS）的结构信息，这将为NRPS的再工程以生产新型抗生素提供信息。主题[3]涉及充分表征“单向”氨基酸消旋酶的动力学和寡聚化性质。虽然一些消旋酶在特定条件下可能看起来是“单向的”，但它们不太可能是真正的单向的。我们将开发一个框架来理解这种行为的基础上的动力学参数和/或这些酶的寡聚化特性。主题[4]致力于开发一种基于活性的蛋白质谱分析试剂，用于检测生物体中所有蛋白质（蛋白质组）之间的特定活性位点结构。我们已经开发了一种反应性探针，将选择性地标记一个特定的架构，从而允许识别潜在的药物开发目标。该研究计划提供了一个多学科，尊重，协作和包容的培训环境，将有利于各级学员。受训者培养酶动力学，蛋白质化学，物理有机实验室技术，有机合成，微生物学和分子生物学的技能-任何人追求在制药，生物技术或学术界的职业生涯资产。

Enzymes are proteins that accelerate (catalyze) biological reactions. Our research focuses on understanding how catalysis arises from the interactions that occur between enzymes and the substrates they act on. This knowledge is essential to understand how enzymes work, to engineer new enzyme catalysts, and to design enzyme inhibitors (drugs/herbicides). Primarily, we study mandelate racemase (MR), which catalyzes the interconversion of D- and L-mandelic acid and serves as a paradigm for understanding how enzymes catalyze unfavorable reactions (i.e., C-H bond cleavage). Racemases and epimerases are of great interest because of their utility for the preparation of D-amino acids, rare sugars, and other value-added compounds. This proposal extends our research on enzymes through 4 themes. Theme [1] focuses on labelling catalytic acid-base residues of a racemase with NMR-active nuclei to distinguish their protonation states. These studies will furnish direct evidence of how substrate-binding alters the pKa of the acid-base catalysts at the active sites of racemases. This knowledge aids the rational design of novel enzyme catalysts. Theme [2] elaborates two enzyme inhibition strategies we developed: inhibition of enzymes acting on carbon acid substrates by boronic acids and the inhibition of racemases by substrate-product analogues (SPAs). We will examine thermodynamic and structural aspects of inhibition of MR by boronic acids to clarify how boronic acid-based inhibitors interact with such enzymes. The results will be of interest to pharma since the boronic acid group affords a point of molecular recognition on drugs and exhibits low toxicity. We will synthesize an SPA to obtain structural information on a non-ribosomal peptide synthetase (NRPS), which will inform the re-engineering of NRPSs to produce novel antibiotics. Theme [3] involves fully characterizing the kinetic and oligomerization properties of a "unidirectional" amino acid racemase. While some racemases may appear to be "unidirectional" under specific conditions, it is unlikely that they are truly unidirectional. We will develop a framework for understanding this behaviour based on the kinetic parameters and/or oligomerization properties of these enzymes. Theme [4] focuses on developing an activity-based protein profiling reagent for the detection of specific active-site architectures among all the proteins (proteome) in an organism. We have developed a reactive probe that will selectively label a specific architecture, thereby permitting identification of potential targets for drug development. This research program offers a multidisciplinary, respectful, collaborative, and inclusive training environment that will benefit trainees at all levels. Trainees develop skills in enzyme kinetics, protein chemistry, physical organic laboratory techniques, organic synthesis, microbiology, and molecular biology - assets for anyone pursuing a career in pharma, biotechnology, or academia.