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

✍ Scribed by Matthew J. Todd; Javier Gomez

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 119 KB
• Volume: 296
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5218

✦ Synopsis

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.