Enzyme reactions, FABMS

In the determination of steady state reaction kinetic constants of enzyme-substrate reactions, FABMS also provides some very unique capabilities. Since these studies are best performed in the absence of glycerol in the reaction mixture, the preferred method is that which analyzes aliquots which are removed from a batch reaction at timed intervals. Quantitation of the reactants and products of interest is essential. When using internal standards, generally, the closer in mass the ion of interest is to that of the internal standard, the better is the quantitative accuracy. Using these techniques in the determination of kinetic constants of trypsin with several peptide substrates, it was found that these constants could be easily measured (8). FABMS was used to follow the decrease in the reactant substrate and/or the increase in the products with time and with varying concentrations of substrate. Rates of reactions were calculated from these data for each of the several substrate concentrations used and from the Lineweaver-Burk plot, the values of Km and Vmax are obtained. 

A number of recent studies have shown that under certain conditions, FABMS indeed can very accurately measure the balance of ionic species in ongoing chemical reactions in solutions. These studies include the determination of acid dissociation constants (2), equilibrium constants for enzyme catalyzed reactions (1), metal-ligand association constants 03), and measurements of... 

Other types of reactions which have been studied using FABMS include those catalyzed by enzymes. This application is particularly interesting because it represents for the first time a generally useful and molecularly specific probe with which to measure a wide variety of enzyme substrates and products. Two approaches have been successful, one in which the reaction is followed by the removal of aliquots of sample taken at timed intervals with subsequent analysis by FABMS and the other allowing the reaction to take place in a glycerol-water mixture on the probe directly inside the mass spectrometer. The choice of either method depends upon the application. If the prime interest is to analyze a substrate, for example, monitoring the release of amino acids from a polypeptide using an exopeptidase, then direct analysis inside the spectrometer may be preferred. If, on the other hand, the prime interest lies... 

The molecular specificity of FABMS opens new areas for kinetic analysis of enzyme-substrate interactions. Since the method is applicable to virtually all substrates whether or not they have a UV or visible spectrum, natural substrates can be used in place of synthetic substrates. Although much has been learned through use of the latter, their reaction constants can indeed be quite different than those of natural substrates. 

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