Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?
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 Helicobacter 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.
Todd
M J
MJ
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA. mtodd@jhu.edu
Gomez
J
J
D000255:Adenosine Triphosphate / Q000737:chemistry; D002151:Calorimetry / Q000379:methods*; D018834:Chaperonin 60 / Q000032:analysis; D018833:Chaperonins / Q000032:analysis; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry; D005417:Flavobacterium / Q000201:enzymology; D016333:HIV Protease / Q000032:analysis; D016480:Helicobacter pylori / Q000201:enzymology; D006493:Heparin / Q000378:metabolism; D019763:Heparin Lyase / Q000032:analysis; D007700:Kinetics; D020543:Proteome / Q000032:analysis; D014357:Trypsin / Q000032:analysis; D014510:Urease / Q000032:analysis*
D018834:Chaperonin 60; D020543:Proteome; D000255:Adenosine Triphosphate; D006493:Heparin; D014357:Trypsin; D016333:HIV Protease; D014510:Urease; D018833:Chaperonins; D019763:Heparin Lyase