Enzyme kinetics determined using calorimetry: A general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal ti tration microcalorimetry are presented. The methods are based on the propor tionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substra te, while pseudo-first-order conditions are maintained. (ii) Following a si ngle injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characte rization in a single experiment and can be used to measure enzyme inhibitio n. 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 u rease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.2 1.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activ ity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is com pletely general, enabling precise analysis of reactions in spectroscopicall y opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics. (C) 2001 Academic Press .