Reaction of Wild-Type, C365S, and C458SSaccharomyces cerevisiaePhosphoenolpyruvate Carboxykinases with Fluorescent Iodoacetamide Derivatives

The reactivities of Cys365 and Cys458 of ATP-dependentSaccharomyces cerevisiaephosphoenolpyruvate (PEP) carboxykinase against a range of sulfhydryl reagents have been investigated. The effect of pH on the second order reaction constants ofN-(1-pyrenyl)maleimide with mutant C458S and C365S PEP carboxykinases allowed the determination of pKavalues of 9.4 and 9.1 for Cys365 and Cys458, respectively. The analysis of the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microenvironment of these residues is rather polar. Anisotropy measurements and acrylamide quenching experiments carried out withN-(iodoacetyl)-N′-(5-sulfo-1-naphthyl)ethylenediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked to Cys458 than to Cys365. These findings point to differences in the protein microenvironments around Cys365 and Cys458 inS. cerevisiaePEP carboxykinase. A comparison of the results obtained with published data for GTP-dependent PEP carboxykinases, suggest significant differences for the protein region around the reactive cysteinyl residues of these enzymes.