Role of Binding Determinants in Enzyme Catalysis

Enzymes are proteins that accelerate (catalyze) biological reactions. My 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, I 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., CH bond cleavage). Previously, we discovered that MR is inhibited by substrate-product analogues and developed a general design strategy for inhibiting cofactor-independent racemases. The present proposal extends this research program through four themes. Theme [1] addresses the hypothesis that substrate binding modulates the environment of the Brønsted acid-base catalyst Lys 166 of MR (and hence its pKa). A photocaged -15N-Lys will be introduced at position 166. After deprotection, 15N NMR spectroscopy will be used to observe how the binding of substrate analogues changes the 15N chemical shift, thereby indicating how ligand binding alters the environment of the Brønsted acid-base catalysts of racemases. Theme [2] focuses on altering the hydrophobic cavity of MR to change its substrate specificity. Our hypothesis is that an interdigitating loop between adjacent subunits is a prime determinant of substrate specificity between MR and the related enzyme D-tartrate dehydratase (TarD). The tip of this loop (Leu 93) will be mutated to determine the effect on MR catalysis and oligomeric state. Lys at the tip enhances the binding of D-tartrate by MR and this will serve as a starting point for engineering MR into TarD. These studies will provide the “rules” for building substrate specificity into a hydrophobic pocket. Theme [3] explores the role(s) of metal ions in enolase superfamily enzymes. Preliminary studies suggest that MR modulates the effective charge of the bound metal ion. Weakening the MR-metal interactions through mutagenesis will be used to test this hypothesis. The metal ion dependence of TarD will also be explored to discover if the metal ion is essential for catalysis. Theme [4] focuses on developing an activity-based protein profiling agent to identify active site architectures in proteomes. A methyl acyl phosphate-based reagent will be used to identify enzymes containing a nucleophilic site adjacent to a cationic site. This approach should permit identification of families of enzymes in proteomes that share reactive motifs that could be targeted for inhibitor/drug development. Trainees in this multidisciplinary environment develop skills in organic synthesis, protein chemistry, enzyme kinetics, microbiology, and molecular biology assets for anyone interested in pursuing careers in pharma, biotechnology, or academia.