Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458.
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.
Krautwurst
H
H
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago.
Encinas
M V
MV
Marcus
F
F
Latshaw
S P
SP
Kemp
R G
RG
Frey
P A
PA
Cardemil
E
E
D000595:Amino Acid Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine