Enzyme Kinetics Determined Using Calorimetry: A General Assay for Enzyme Activity? 

 -  Analytical BioChemistry, 296(2), p.179-187, 2001 .
<br/><br/> Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time)generated. (i)An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii)Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i)oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii)transferase activity of creatine phosphokinase (EC 2.7.3.2)and hexokinase (EC 2.7.1.1); (iii)hydrolytic activity of Heliobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv)lyase activity of heparinase (EC 4.1.1.7); and (v)ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.