Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: Revised Amino Acid Sequence, Site-Directed Mutagenesis, and Microenvironment Characteristics of Cysteines 365 and 458

Abstract

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

Physical and Genetic Interactions of Cytosolic Malate Dehydrogenase with Other Gluconeogenic Enzymes

“…To construct conventional two-hybrid vectors (pASPCK1 and pACT-PCK1) containing PCK1 sequences, PCR was conducted with a YEp352 plasmid carrying PCK1 (provided by Dr. E. Cardemil) (25) to generate the PCK1 coding region flanked by EcoRI restriction sites for subcloning. Similar vectors (pASFBP1 and pACTFBP1) containing FBP1 sequences were generated using PCR with yeast genomic DNA as the template to generate the FBP1 coding region with 5Ј and 3Ј XhoI restriction sites for ligation into plasmid SalI or XhoI sites.…”

Isolation and nucleotide sequence of the gene encoding phosphoenolpyruvate carboxykinase fromKluyveromyces lactis

“…Genomic Southern hybridization (Southern, 1975) with the ECL direct nucleic acid labelling and detection systems (ECL system; Amersham International plc) and colony hybridization (Walter and Holtke, 1992) with DIG-High Prime DNA labelling and detection kit I (Boehringer Mannheim) was performed for cloning the KlPCK1 gene with PCK1, the PEPCK gene of S. cerevisiae, as the probe. PCK1 was amplified by polymerase chain reaction (PCR; Sambrook et al, 1989) using genomic DNA of S. cerevisiae YNN27 as a template and two primers selected to hybridize with the 5 and 3 ends of the Pck1 ORF (Krautwurst et al, 1995). Sequencing was performed on the deletion mutants prepared using a Kilo-Sequence Deletion Kit (Takara Shuzo).…”

“…A mutant of Saccharomyces cerevisiae lacking PEPCK, has been isolated (Perea and Gancedo, 1982). The PCK1 gene coding PEPCK has been cloned by functional complementation of the mutant (Valdés-Hevia et al, 1989) and sequenced (Stucka et al, 1988;Krautwurst et al, 1995). The expression of the PCK1 gene in S. cerevisiae is strictly regulated and dependent on the carbon source provided (Proft et al, 1995).…”

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

“…The fractions corresponding to each peak were concentrated and sequenced, and Table II shows the Edman degradation results for both peptides. Peptide 2 is comprised of amino acid residues Asn-359 to Val-374, a peptide containing the reactive Cys-365 (Krautwurst et al, 1995). In cycle 7 a signal appeared at the same position as PTH-carboxymethylcysteine.…”

“…PEPCK was obtained according to Krautwurst et al (1995). The rate of oxaloacetate formation was determined spectrophotometrically at 30~ as described by Malebrfin and Cardemil (1987) for the yeast enzyme and Bazaes et al (1993) for the bacterial PEPCK.…”