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Structural and catalytic characteristics of Escherichia coli adenylate kinase.

1987; Elsevier BV; Volume: 262; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(19)75829-3

1083-351X

Isabelle Saint Girons, Anne‐Marie Gilles, Danielle Margarita, S. Michelson, Monique Monnot, Serge Fermandjian, Antoine Danchin, Octavian Bârzu,

Enzyme Structure and Function

Biochike, CEN de Saclay; Gif-sur-Yvette, FrinceThe 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 singlestep purification procedure.Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the Nterminal 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 Acide 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-&~-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.Adenylate kinase (EC 2.7.4.3) is the smallest phosphotransferase.Muscle enzyme (myokinase) has been purified and crystallized 30 years ago (1).Since then, the adenylate kinase from different sources was isolated in pure form (2-16) and investigated extensively using various physicochemical approaches (17-23).Comparison of the primary structure of five types of muscle adenylate kinase (pig, human, calf, chicken, and rabbit) determined either by protein sequencing or deduced from cDNA nucleotide sequencing revealed an extremely high degree of homology (15, 24).Adenylate kinase from lower eukaryotes and prokaryotes has not been characterized as thoroughly.The enzyme from yeast, Paracoccus denitrificans, and Escherichia coli has been purified, and the kinetic properties have been compared with