| Abstract: | A simple method to identify functional amino acids in enzymes is described. This method is based on the mass spectrometric detection of molecular weight changes as the consequence of chemical modification of enzymes with group-specific reagents. Here we report the use of phenylglyoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyrocarbonate to identify functional amino acid residues. Precise information is obtained about the stoichiometry of reaction, and a relationship between the loss of enzyme activity and the amount of chemical modification is easily established. Modification sites are located by proteolytic digestion of the modified enzyme, followed by peptide mapping based on high-pressure liquid chromatography using an electrospray mass spectrometer as an on-line detector. In comparison with more conventional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodology is limited only by the stability of the chemically modified species produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have been confirmed by site-directed mutagenesis and X-ray crystallography. |
