REACTION OF WILD-TYPE, C365S, AND C458S SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASES WITH FLUORESCENT LODOACETAMIDE DERIVATIVES

The reactivities of Cys365 and Cys458 of ATP-dependent Saccharomyces c erevisiae phosphoenolpyruvate (PEP) carboxykinase against a range of s ulfhydryl reagents have been investigated. The effect of pH on the sec ond order reaction constants of N-(1-pyrenyl)maleimide with mutant C45 8S and C365S PEP carboxykinases allowed the determination of pK(a) val ues of 9.4 and 9,1 for Cys365 and Cys458, respectively, The analysis o f the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microe nvironment of these residues is rather polar, Anisotropy measurements and acrylamide quenching experiments carried out with N-(iodoacetyl)-N '-(5-sulfo-1-naphthyl)et enediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked t o Cys458 than to Cys365. These findings point to differences in the pr otein microenvironments around Cys365 and Cys458 in S. cerevisiae PEP carboxykinase. A comparison of the results obtained with published dat a for GTP-dependent PEP carboxykinases, suggest significant difference s for the protein region around the reactive cysteinyl residues of the se enzymes. (C) 1996 Academic Press, Inc.