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 (k_cat  = 1.5 x 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. From a catalysis perspective these two examples suggest a new and potentially powerful strategy for enzyme engineering, the rational design of intermolecular interaction outside of an enzyme’s active site that enhance and control enzyme kinetics.

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 (K_M) 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 co-factor binding with an alcohol dehydrogenase (AdhD) through rationally designed molecular interactions between the NAD⁺ 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 nearly 3-fold decrease in K_M,app resulting in a similar increase in enzyme efficiency (k_cat/K_M). In the case of AdhD we were able to reduce co-factor binding by nearly one half. These findings represent an important first step towards developing a new strategy of enzyme engineering by means of controlled substrate-enzyme interactions.