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 substra...

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Bibliographic Details
Published in:Analytical biochemistry 2001-09, Vol.296 (2), p.179-187
Main Authors: Todd, Matthew J., Gomez, Javier
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - chemistry
Calorimetry - methods
Chaperonin 60 - analysis
Chaperonins - analysis
DNA Mutational Analysis
Escherichia coli - chemistry
Flavobacterium - enzymology
Helicobacter pylori - enzymology
Heparin - metabolism
Heparin Lyase - analysis
HIV Protease - analysis
Kinetics
Proteome - analysis
Trypsin - analysis
Urease - analysis
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2001.5218