Analysis of progress curves for enzyme-catalyzed reactions: application to unstable enzymes, coupled reactions and transient-state kinetics.
There are several advantages to the use of progress curves to analyze the the kinetic properties of enzymes but most studies still rely on rate measurements. One of the reasons for this may be that progress curve analysis relies on the enzyme and the reactants being completely stable under assay conditions. Here a method is described that relaxes this requirement and allows progress curve analysis to be applied to unstable enzymes. The procedure is based on a combination of numerical integration and non-linear regression to fit rate equations to the progress curve data. The analysis is verified using simulated data and illustrated by application to the reaction catalyzed by alkaline phosphatase, measured in the presence of 10 mM EGTA where it has a half-life of 3 1/2 min. The method may also be applied to other experimental systems where the development over time reveals important properties but where an analytical solution of the underlying model is not known. This extension is illustrated by two systems: the coupled reactions catalyzed by pyruvate kinase and lactate dehydrogenase under conditions where both enzymes have similar activity; and the transient-state kinetics of the reaction catalyzed by glutamate dehydrogenase.
Duggleby
R G
RG
Department of Biochemistry, University of Queensland, Brisbane, Australia.
D000469:Alkaline Phosphatase / Q000378:metabolism; D003198:Computer Simulation; D004795:Enzyme Stability; D004798:Enzymes / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D012984:Software
D004798:Enzymes; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D009243:NAD; C008644:nitrophenylphosphate; D009249:NADP; D007770:L-Lactate Dehydrogenase; D005969:Glutamate Dehydrogenase; D011770:Pyruvate Kinase; D000469:Alkaline Phosphatase