Targeting enzyme sites in pyruvate kinase, glutamate dehydrogenase and glutathione S-transferase

Abstract

Purine nucleotide analogs with reactive functional groups at various positions of the purine or ribose ring can be effective in mapping the active sites and regulatory sites of kinases and dehydrogenases. We have synthesized 5’-p-fluorosulfonylbenzoyl (FSB) derivatives of adenosine, guanosine, 8-azidoadenosine and the fluorescent l, N^-ethenoadenosine in which the electrophilic FSB moiety occupies the pyrophosphate region of the natural nucleotide. In addition, analogs with a 4-bromo-2, 3-dioxobutylthio (BDB-T) moiety adjacent to the 2, 6 or 8 positions of the purine ring of nucleotides have been synthesized. These compounds bind to specific nucleotide sites in pyruvate kinase, glutamate dehydrogenase and other enzymes prior to covalently labeling those sites at amino acids such as cysteine, tyrosine, lysine, histidine, aspartic acid and glutamic acid. Affinity labeling experiments have been used directly to probe structure-function relationships in enzymes, as well as to rationally select targets for site-directed mutagenesis. We have also synthesized a reactive bromodioxobutyl derivative of the tripeptide glutathione. Studies of pyruvate kinase, glutamate dehydrogenase and glutathione S-transferase are described in this paper which illustrate the application of these compounds to affinity labeling of enzymic catalytic sites. © 1994 IUPAC