Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. By using microcalorimetry, the thermokinetic method was developed to kinetic studies on Na+/K+-ATPase for the first time. Compared with other ones, the method provided accurate measurements of not only thermodynamic data but also the kinetic data. At 310.15 K and pH=7.4, the molar reaction enthalpy rHm was measured as (-40.408±1.9) kJ·mol-1. The Michaelis constant Km was determined to be (0.479±0.020)×10-3 mol·L-1 and consistent with literature figure which is about 0.5×10-3 mol·L-1. The maximum velocity Vmax obtained was (0.681±0.026) 靘ol Pi·min-1·mg protein-1. All of the data have good repeatability and self-consistency. The reliability of thermokinetic method was verified by the experimental results and further confirmed by colorimetric studies. Moreover, the effect of enzyme pre-dilution on its activities was also investigated.
A new thermokinetic reduced extent method for studying of the reversible competitive inhibition of single sub-strate enzyme-catalyzed reactions was proposed in this paper. The reaction that arginase-catalyzed hydrolysis of L-arginine to L-ornithine and urea and the inhibition of this reaction by the product, L-ornithine, and exogenous L-lysine were studied at 37 ℃ in 40 mmolL-1 sodium barbiturate-HCl buffer solution (pH=9.4). Michealis con-stant Km for arginine and maximum velocity Vm of the reaction were determined to be 5.14 mmolL-1 and 1.13× 10-2 mmolL-1s-1, respectively. The product inhibition constant KP and inhibitory constant KI of L-lysine were de-termined to be 1.18 and 5.6 mmolL-1, respectively. All the results have better repeatability and self-consistency and are in agreement with literature values. This new method using more direct thermal information from the proc-ess would give more reliable kinetic information than the traditional initial rate method.
