IDENTIFICATION OF REACTIVE VICINAL CYSTEINES IN SACCHAROMYCES-CEREVISIAE (ATP) AND SYSTOLIC RAT-LIVER (GTP) PHOSPHOENOLPYRUVATE CARBOXYKINASES

Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phosphoen olpyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1 -pyrenyl)-iodoacetamide. Reagent incorporation was completely prevente d by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subu nit were incorporated. The fluorescence spectra of the labeled protein s showed the pyrene excimer emission band. The pyrenyl-derivatized enz ymes were digested with trypsin after carboxymethylation, and two labe led peptides were isolated for each carboxykinase upon reverse-phase h igh-performance liquid chromatography. Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzym e), and cysteines 288 and 413 (rat enzyme) were labeled with the fluor escence SH-specific reagent. The relative reactivity of these residues was characterized. Labeling experiments utilizing the 5,5'-dithiobis( 2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-group s occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region.