Role of Binding Determinants in Enzyme Catalysis

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.