371767 Controlling Local Substrate Concentrations and Enzyme Kinetics through Rationally Designed Intermolecular Interactions
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. Mutations to SOD that increase the local substrate-enzyme interactions have been shown to push catalysis faster than diffusion-limited rates. 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. Browian dynamics simulations of this system are in agreement with our experimental data and demonstrate increased local substrate concentrations due to molecular interactions between the substrate and DNA attached to HRP. 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. Using this system, we also vary the location of the DNA conjugation site to explore the effects of DNA location of kinetic enhancements and increased local substrate concentrations. In the case of HRP we achieve a 3-fold decrease in KM,app resulting in a similar increase in enzyme efficiency (kcat/KM). In the case of AdhD we were able to reduce KM,app by more than one half. These findings represent an important first step towards developing a new strategy of enzyme engineering by means of controlled substrate-enzyme interactions.
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