J. Biochem, 1986, Vol. 99, No. 5 1299-1310
© 1986 Japanese Biochemical Society
research-article |
Phosphoenolpyruvate Carboxylase of Escherichia coli K-12. N- and C-Terminal Sequences and Tentative Assignment of the Catalytically Essential Cysteine Residue1
*Department of Chemistry, Faculty of Science, Kyoto University Sakyo-ku, Kyoto 606
**College of Liberal Arts, Kinki University Higashiosaka, Osaka 577
The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31 [EC] ] 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-(dimethylamion)-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. Further more, 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.
1This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan, the Nissan Science Foundation, and Sumitomo Chemical Co., Ltd.