Enzyme kinetics determined by single-injection isothermal titration calorimetry

Abstract

The purposes of this paper are (a) to examine the effect of calorimeter time constant (τ) on heat rate data from a single enzyme injection into substrate in an isothermal titration calorimeter (ITC), (b) to provide information that can be used to predict the optimum experimental conditions for determining the rate constant (k2), Michaelis constant (KM), and enthalpy change of the reaction (ΔRH), and (c) to describe methods for evaluating these parameters. We find that KM, k2 and ΔRH can be accurately estimated without correcting for the calorimeter time constant, τ, if (k2E/KM), where E is the total active enzyme concentration, is between 0.1/τ and 1/τ and the reaction goes to at least 99% completion. If experimental conditions are outside this domain and no correction is made for τ, errors in the inferred parameters quickly become unreasonable. A method for fitting single-injection data to the Michaelis–Menten or Briggs–Haldane model to simultaneously evaluate KM, k2, ΔRH, and τ is described and validated with experimental data. All four of these parameters can be accurately inferred provided the reaction time constant (k2E/KM) is larger than 1/τ and the data include enzyme saturated conditions.