Enzyme kinetics monitoring

Kettling, U., Koltermann, A., Schwille, P., and Eigen, M. (1998) Real time enzyme kinetics monitored by dualcolor fluorescence cros-correlation spectroscopy, Proc. Natl. Acad. Sci. 95, 1416-1420. 

Ramanathan M, Simonian AL (2007) Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy application for neurotoxins detection. Biosens Bioelectron 22 3001-3007... 

With a considerable increase in the number of reflections (only three were used in the experiments mentioned above) and an increase in laser power the signal to noise ratio could perhaps be improved enough to allow monitoring of the enzyme kinetics by means of time-resolved spectroscopy. 

NMR can help to monitor energization, see, e.g. [121, 273], especially the levels of 31P-containing metabolites, e.g. [45, 366], enzyme kinetics, compartmentalized intracellular ion activities, the fate of 3H-, 2H-, 13C-, 15N-, or 19F-labeled tracers, e.g. [108,109], 02 tension, compartmentalized redox potential, membrane potential, cell number or cell volume, see [133], and even pH. Major drawbacks are the cost of the equipment, the low intrinsic sensitivity and the interpretation of spectra [430]. 

Thus, as the first approach to kinetic studies of the action of /8-D-glu-cosiduronase toward synthetically prepared 1-O-acyl-a- and -/3-D-glu-copyranuronic acids, a reliable method that would permit monitoring of the rate of enzymic hydrolysis was needed. For this purpose, To-masic and Keglevic268 developed an analytical procedure, involving the colorimetric reaction of D-glucuronic acid with benzidine in the presence of acetic acid,269 that proved to be fully applicable to enzymic, kinetic studies performed with 1-esters of D-glucuronic acids and with D-glucosiduronic acids as the substrates. 

The means by which enzymes facilitate the homolytic cleavage of the Co-C5 bond has been addressed in detail in studies of MCM. This process can be kinetically monitored by the spectral change from Co(III) in coenzyme B12 to that of cob(II)alamin. This spectral change depends on the addition of the substrate to the complex of MCM and adenosylcobalamin. The kinetic barrier to bond cleavage is lowered by 17 kcalmol . " " Moreover, the rate of this change displays a kinetic isotope effect of >20 (Fh/F ) when the deuterated substrate is employed. " It was concluded that Co-C5 bond cleavage and hydrogen abstraction from the substrate are kinetically coupled. This effect has been reported for other coenzyme Bj2-dependent reactions as well. 

FIGURE 15.13 Analysis of enzyme kinetics of 5-warfaiin 7-hydroxylase (CYP2C9) by QTRAP. (a) (A /Fmax) determination for the formation of 7-hydroxy-5-warfarin from 5-warfarin in HLM (7-hydroxy-5-warfarin was monitored with MRM transition from m/z 325>179). (p) IC50 determination for inhibition of CYP2C9 by sulfaphenazole. 

ESI Study of enzyme kinetics, inhibition, multiple reaction monitoring Norris ef al. [52]... 

Norris, A.J., Whitelegge, J.P., Faull, K.F., Toyokuni, T. (2001) Analysis of Enzyme Kinetics Using Electrospray Ionization Mass Spectrometry and Multiple Reaction Monitoring Fucosyltransferase V. Biochemistry 40 3774-3779. 

Site-directed mutagenesis is an indispensable technique for determining the effect of substituting a specific amino acid writh another. For example, enzyme reaction rates can be measured for both wdld type and mutant enzymes, and changes in enzyme kinetics can be monitored to assess the possible catalytic role of a given residue. The complete absence of activity in a mutant enzyme indicates that the mutated residue is essential for catalysis. 

A frequent result of an enzymatic reaction is a pH-change that may be monitored by a pH-sensitive i.s.e. In such cases, a major interfer-ant is the buffer capacity, which is essential for optimal function of the biological system used (e.g. enzymatic reaction) [68-70]. The enzyme layer thickness [71], enzyme loading and enzyme kinetics play important roles in both the sensitivity and dynamic range of these sensors. 

Experimental studies of enzyme kinetics are typically conducted by monitoring the initial rate of product formation in a solution in which the enzyme is present at very low concentration. Indeed, enzymes are such efficient catalysts that significant accelerations may be observed even when their concentrations are more than three orders of magnitude smaller than those of their substrates. 

Enzyme kinetics Reaction monitoring, proton transfer... 

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