CHEMICAL MODIFICATION MONITORED BY ELECTROSPRAY MASS-SPECTROMETRY - ARAPID AND SIMPLE METHOD FOR IDENTIFYING AND STUDYING FUNCTIONAL RESIDUES IN ENZYMES

A simple method to identify functional amino acids in enzymes is descr ibed. This method is based on the mass spectrometric detection of mole cular weight changes as the consequence of chemical modification of en zymes with group-specific reagents. Here we report the use of phenylgl yoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyr ocarbonate to identify functional amino acid residues. Precise informa tion is obtained about the stoichiometry of reaction, and a relationsh ip between the loss of enzyme activity and the amount of chemical modi fication is easily established. Modification sites are located by prot eolytic digestion of the modified enzyme, followed by peptide mapping based on high-pressure liquid chromatography using an electrospray mas s spectrometer as an on-line detector. In comparison with more convent ional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodo logy is limited only by the stability of the chemically modified speci es produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have b een confirmed by site-directed mutagenesis and X-ray crystallography. (C) Munksgaard 1998.