Enzyme Engineering via Rationally Designed Intermolecular Interactions: Applications towards Bioelectrocatalysis

Wednesday, 8 October 2014: 08:00
Expo Center, 2nd Floor, Beta Room (Moon Palace Resort)
Y. Gao, J. Zhu, J. L. Lin, and I. Wheeldon (University of California, Riverside)
In nature we find many examples of enzymes and multienzyme stuctures where catalysis is enhanced by well-designed molecular interactions between the enzymes and their substrates. Two compelling examples are the enzyme superoxide dismutase (SOD) and the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR). SOD, one of the fastest known enzymes (kcat  » 1.5 ´ 109 M-1s-1), uses charge complementarity to produce substrate-enzyme interactions that enhance enzyme kinetics by directing the substrate to the enzyme’s active site. A positively charge patch on the surface TS-DHFR restricts diffusion of a negatively charged reaction intermediate to a pre-defined channel between two active sites, sequestering the intermediate along the enzyme’s surface and preventing diffusion to the bulk. This bounded diffusion promotes substrate channeling, enhancing pathway catalysis by protecting the intermediate from undesired side reactions. These examples are informative: They suggest that rational design of substrate-enzyme interactions can be used to enhance enzyme catalysis. In this work we engineer new enzyme structures with quantifiable binding interactions between the enzyme and its substrate. We hypothesize that the engineered molecular interactions will lead to increases in local substrate concentrations thereby enhancing enzyme catalysis. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant (KM) of horseradish peroxidase (HRP) modified with a double stranded DNA structure that exhibits sequence dependent binding of phenolic HRP substrates. We extend this work to a second experimental system and demonstrate enhanced catalysis with an alcohol dehydrogenase (AdhD) through rationally designed molecular interactions between the NAD+ co-factor mimic nicotinamide mononucleotide (NMN+) and a double stranded DNA structure conjugated near the enzyme’s active site. Demonstration of rationally designed kinetic enhancements with enzyme co-factors such as NMN+ is an important demonstration with respect to bioelectrocatalysis as many such systems rely on this type of co-factor.  We aim to extend this work towards other co-factor and substrates relevant to electrochemical biosensor and enzymatic biofuel cells.