Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC.

Abstract

There is growing interest in using isothermal titration calorimetry (ITC) to characterize enzyme kinetics by measuring the heat produced or absorbed by catalysis in real time. Since virtually all chemical reactions are associated with changes in enthalpy, ITC represents a robust and nearly universal experimental approach. Nevertheless, there are technical challenges that limit ITC's applicability. For instance, the full kinetic characterization of enzymes with two substrates (bi-substrate enzymes), which comprise the majority of known examples, requires a series of experiments to be performed as the concentrations of both substrates are varied. This is a time-consuming and expensive process using current ITC methods since many (>5) individual experiments must be performed independently to obtain a sufficient quantity of data. We have developed a new ITC method, which we term 2D-ITC, which maps the reaction velocity as a function of two substrate concentrations in a single, roughly 2 h long experiment. This method provides a level of detail that rivals or exceeds any existing enzyme assay, as a single experiment generates on the order of 7000 catalytic rate measurements. In a proof-of-principle application to rabbit muscle pyruvate kinase (rMPK), the method correctly identified the enzyme's random sequential mechanism and allosteric catalytic suppression by the amino acid phenylalanine (Phe). Unexpectedly, we found that while Phe reduces affinity for the substrate phosphoenolpyruvate, a known phenomenon, it also alleviates inhibition by the reaction product ATP, which had not been reported previously. Given the relative abundance of ATP in the cell, this opposing effect is expected to have a substantial impact on rMPK activity.