PMID:3027060
| Citation |
Saint Girons, I, Gilles, AM, Margarita, D, Michelson, S, Monnot, M, Fermandjian, S, Danchin, A and Bârzu, O (1987) Structural and catalytic characteristics of Escherichia coli adenylate kinase. J. Biol. Chem. 262:622-9 |
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| Abstract |
The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate. |
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| Keywords |
Adenylate Kinase/genetics; Adenylate Kinase/isolation & purification; Adenylate Kinase/metabolism; Amino Acid Sequence; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Genes; Genes, Bacterial; Kinetics; Phosphotransferases/genetics; Plasmids; Substrate Specificity |
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