An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe

An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2 ',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K-m, K-i, k(c), and DeltaH(m) can be determined by two simple calorimet ric measurements. First, the substrate was titrated into the cell containin g high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations o f product can be calculated from the peak height and the molar reaction hea t. From Michaelis-Menten function in the presence of inhibitors, the relati onship between K-m and K-i can be obtained. Then, the dissociation constant , which is equal to K-i, was measured by a regular ITC experiment. Thus, K- m and k(c) can be calculated. The method developed here can be applied in o ther enzyme catalytic systems with inhibitive products. (C) 2001 Elsevier S cience.