UC Riverside

Enzymes are highly effective catalysts that enable biological reactions and control complex metabolic and cell processes. Over the past few decades, active site engineering has dramatically improved enzyme kinetics; however, in many cases catalytic efficiency (kcat/KM) can be limiting to the desired application. There have also been considerable efforts put towards increasing enzyme stability through immobilization on solid supports. Recently, nanoscale molecular engineering tools have opened the possibility of replicating the enhancement of immobilization while also controlling an enzyme’s local chemical and physical microenvironment. These new molecular tools have opened a new approach to enzyme engineering that has the potential to both control enzyme kinetics as well as provide the benefits of increased stability often observed with immobilization. Here, we propose the design and application of rationally designed biomolecular scaffolds to control Rationally Designed Scaffold Increase Enzyme and Cascade Kinetics via Local Environmental iv an enzyme’s microenvironment and consequently enhance reaction kinetics. The first example of this approach is the engineering of phosphotriesterase (PTE), a potential therapeutic countermeasure for organophosphate toxicity, with DNA scaffolds. The challenge is that highly toxic nerve agents, such as VX or Sarin, are potentially deadly at very low concentration, thus requiring a biocatalytic scavenger with high catalytic rates at low concentrations. We show that small DNA scaffolds designed to bind organophosphates in a sequence-dependent manner can decrease the Michaels parameter (KM) and increase the second order rate constant of PTE by nearly 11-fold, thus providing high catalytic rates at low organophosphate concentrations. We also show that the position of the scaffold is critical to the enhancement; conjugation of the scaffold close to the active site provides the largest enhancement provided that the active site is not blocked by the appended scaffold. The second example of our approach is to control a microenvironment of a two- step enzyme cascade. We modeled the cascade reaction between hexokinase-2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD) and linked them by superfold green fluorescent protein (sfGFP) with different charges (+36, 0, -30). We observed that (+36)sfGFP enhanced cascade kinetics, (-30)sfGFP did the opposite, and (0)sfGFP had no effect on the cascade. We also showed that the modification of sfGFP significantly altered the kinetics of the assembled enzymes, including kcat, KM, and Ki. We simulated and fitted the experiments with the model, and the results showed that the enhanced kinetics of this cascade was due to change in the kinetics of the assembled enzymes, influenced by the microenvironment change of the scaffold.