Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue.
The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000302:isolation & purification; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines; D007773:Lactates; D008301:Maleimides; D010446:Peptide Fragments / Q000032:analysis; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification; D014357:Trypsin
D006898:Hydroxylamines; D007773:Lactates; D008301:Maleimides; D010446:Peptide Fragments; C048984:2-phospholactic acid; D019811:Hydroxylamine; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D014357:Trypsin; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D003545:Cysteine