Publications
Probing Small-Molecule Degradation to Counter Enzyme Promiscuity
Enzymes that degrade specific small molecules could save lives by neutralizing threats from chemical agents in the blood or environment, or by starving pathogenic cells, but promiscuous interactions with other molecules typically limit their effectiveness by blocking the enzyme active site. An obvious solution would be to re-engineer the enzyme to enhance catalytic fidelity, but lack of understanding about how enzymes discriminate between molecules remains a formidable challenge to this approach. Our recent work in collaboration with the University of Texas (UT) suggested a new approach and a model system for understanding enzyme specificity. Asparaginase enzymes catalyze degradation of asparagine, which forms the basis of a medical treatment. Com- petition by the abundant and chemically similar molecule, glutamine, interferes with asparagine decomposition, thus hindering enzyme efficacy. Asparaginase is advantageous as a model degra- dation enzyme because variants that demonstrate different binding affinities and catalytic rates can be compared. Here, we leveraged Sandia and the University of Maryland's strengths in molecu- lar simulation, and UT experimental expertise in asparaginase modification and functional assays, to understand asparaginase specificity. Our results advanced a new hypothesis about asparagi- nase catalytic mechanism that explains for the first time why proximity between the substrate's alpha-carboxyl and carboxamide is absolutely required for catalysis. Based on those insights, we developed the first mutant (Q59L) asparaginase from E. coli that lacks activity toward glutamine. We used that mutant to show that glutaminase activity is required to kill cancer cells that have asparagine synthetase enzymes (ASNS), but not ASNS-negative cancer cells.