SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASE - REVISED AMINO-ACID-SEQUENCE, SITE-DIRECTED MUTAGENESIS, AND MICROENVIRONMENT CHARACTERISTICS OF CYSTEINE-365 AND CYSTEINE-458

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphospho rylating), EC 4.1.1.49] the modification of which leads to enzyme inac tivation have been subjected to site-directed mutagenesis. PEP carboxy kinase is inactivated by alkylation of Cys(365) Or Cys(458); however, mutation of either or both of these residues to serine has little effe ct on the enzymatic activity. These results eliminate any possible cat alytic function for these cysteinyl residues. In the course of this wo rk, discrepancies in the published nucleotide sequence of the S. cerev isiae PEP carboxykinase gene were detected that alter the deduced amin o acid sequence, Several of these descrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the posit ions previously assigned to Cys(364) and Cys(457) correspond to Cys(36 5) and Cys(458). The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by th eir selective modification with the fluorescent reagent N-(1-pyrenyl)m aleimide (PyM). Our findings indicate that Cys(458) is 7-fold more rea ctive toward the sulfhydryl-directed probe than Cys(365), while quench ing experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latte r.