Titration calorimetry applied to the study of thermokinetics - II. Thermokinetics of single-substrate enzyme-catalyzed reactions

Titration calorimetry is emerging as an important tool for characterizing i nteractions of biological macromolecules by virtue of its general applicabi lity, high accuracy and precision. In this paper, two mathematical models f or thermokinetics of a single-substrate enzyme-catalyzed reaction in titrat ion period and in the stopped-titration reaction period, respectively, have been developed, by using titration calorimetry. On the basis of the titrat ion calorimetric curve, one can use these two models to calculate not only the thermodynamic data (Delta(r)H(m)) but also the kinetic data (K-m and k( 2)) for the reaction. Thermokinetics of a cell-studied single-substrate enz ymatic reaction, the catalase-catalyzed decomposition of hydrogen peroxide, was thus investigated by titration calorimetry, and the molar enthalpy (De lta(r)H(m)) was found to be ( -86.75 +/- 0.88) kJ . mol(-1). The Michaelis constant (K-m) for H2O2 and the turn - over number of the enzyme (k(2)) wer e determined by the titration-period thermokinetic model to be (5.41 +/- 0. 24) x 10(-3) mol . L-1 and (3.58 +/- 0.53) x 10(3) s(-1), respectively, whe reas the corresponding kinetic parameters calculated by the stopped-titrati on-reaction-period thermokinetic model were (5.43 +/- 0.21) x 10(-3) mol . L-1 and (3.60 +/- 0.41) x 10(3) s(-1), respectively, at 298.15 K and pH 7.0 . Reliability of the above thermokinetic models was verified by the experim ental data.