Product inhibition study on carbonic anhydrase using spectroscopy and calorimetry.

Kinetic and thermodynamic studies have been made on the effect of the p-nitrophenol product on the activity of bovine carbonic anhydrase in 50 mM Tris buffer pH 7.5, at 300K using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for p-nitrophenol as a product of the enzymatic reaction. A graphical fitting method was used for determination of the binding constant and enthalpy of inhibitor binding using ITC data. The dissociation binding constant was 0.10mM by the microcalorimetric method, which is in good agreement with the value of 0.11mM for the inhibition constant obtained from the spectrophotometric method.