Tuning Enzyme Kinetics through Designed Intermolecular Interactions Far from the Active Site

Abstract

Enzyme–DNA nanostructures were designed to introduce new substrate–enzyme interactions into their reactions, which altered enzyme kinetics in a predictable manner. The designed enzymes demonstrate a new strategy of enzyme engineering based on the rational design of intermolecular interactions outside of the active site that enhance and control enzyme kinetics. Binding interactions between tetramethylbenzidine and DNA attached to horseradish peroxidase (HRP) resulted in a reduced Michaelis constant (KM) for the substrate. The enhancement increased with stronger interactions in the micromolar range, resulting in a 2.6 fold increase in kcat/KM. The inhibition effect of 4-nitrobenzoic hydrazide on HRP was also significantly enhanced by tuning the binding to HRP–DNA. Lastly, binding of a nicotinamide adenine dinucleotide (NAD(H)) cofactor mimic, nicotinamide mononucleotide (NMN(H)), to an aldo-keto reductase (AdhD) was enhanced by introducing NMN(H)–DNA interactions.