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.

Application of 2D-ITC to the Elucidation of the Enzymatic Mechanism of N-Acetylmuramic Acid/N-Acetylglucosamine Kinase (AmgK) from Pseudomonas aeruginosa

“…For DFs on the order of 23 000, the critical F value is 1.042, meaning that any difference in SSE of >4.2% is enough to deem the fits significantly different at a p-value of 0.001. 14,17 In our case, the difference between the best-fitting mechanism (SSE = 70) and the closest contender (random-sequential; SSE = 113) is >60%. Thus, the enzyme mechanism is elucidated with a high degree of confidence.…”

“…As the degrees of freedom increase, the difference between the SSE required for ascertaining better data fitness to one model over the other decreases, because normalS normalS normalE 1 × normalD normalF 2 normalS normalS normalE 2 × normalD normalF 1 follows an F -distribution. For DFs on the order of 23 000, the critical F value is 1.042, meaning that any difference in SSE of >4.2% is enough to deem the fits significantly different at a p -value of 0.001. , In our case, the difference between the best-fitting mechanism (SSE = 70) and the closest contender (random-sequential; SSE = 113) is >60%. Thus, the enzyme mechanism is elucidated with a high degree of confidence.…”

“…Wang and Mittermaier recently described the use of ITC for the extraction of enzymatic kinetic parameters in a single experiment. 14 This versatile 2D-ITC method allowed us to obtain steady-state kinetic parameters of AmgK from a recurring single-injection pattern. Eighteen consecutive 1 μL injections of either NAM or NAG (160 mM) were introduced The interval between the consecutive injections was at least 1200 s to allow complete consumption of the sugar substrate, which was indicated by the return of the thermogram to the baseline.…”

“…Wang and Mittermaier recently described the use of ITC for the extraction of enzymatic kinetic parameters in a single experiment . This versatile 2D-ITC method allowed us to obtain steady-state kinetic parameters of AmgK from a recurring single-injection pattern.…”

“…12,13 In the interest of catalytic characterization of this enzyme, we utilize a recently reported two-dimensional isothermal titration calorimetry (2D-ITC) method. 14 A single 2D-ITC experiment furnishes the full set of steady-state kinetic parameters for a bisubstrate enzyme. The validation of these results is provided by standard methodologies.…”