The amino acid sequence of adenylate kinase from Paracoccus denitrificans and its relationship to mitochondrial and microbial adenylate kinases.
The complete amino acid sequence of adenylate kinase (MgATP + AMP in equilibrium MgADP + ADP) from Paracoccus denitrificans has been determined. 1. The S-[14C]carboxymethylated protein was cleaved with clostripain, cyanogen bromide and endoproteinase Lys-C; 18, 9 and 6 fragments, respectively, were analyzed. Some of these peptides were further degraded by trypsin, Staphylococcus aureus V8 protease and carboxypeptidases A and B. The fragments were separated by HPLC and sequenced with a gas-phase sequencer. 2. Sequencing the whole unblocked protein yielded the N-terminal region. The C-terminal residues were obtained by carboxypeptidase-Y digestion in agreement with the sequence of tryptic and cyanogen bromide peptides. 3. The final sequence shows 217 amino acids with Mr = 23,609 and contains one free cysteine and a disulfide bond. 4. The comparison of the P. denitrificans sequence with other known adenylate kinases shows highest similarity with the structurally known Escherichia coli enzyme (47%). The only and catalytically relevant His in the paracoccal enzyme is close to the site of binding of adenosine(5')pentaphospho(5')adenosine to E. coli adenylate kinase. The disulfide bridge is located in the 30-residue segment, which is indicative of the large variants and is absent in cytosolic adenylate kinase. The similarity to the mitochondrial intermembrane-space and matrix adenylate kinase isoenzymes is only 40% and 30%, respectively, while 39% of redidues are identical to those of yeast cytosolic adenylate kinase. Therefore, adenylate kinases do not support the hypothesis of a close relationship between Paracoccus and mitochondria.
Spürgin
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P
Institut für Organische Chemie und Biochemie Universität Freiburg im Breisgau, Federal Republic of Germany.
Tomasselli
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AG
Schiltz
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E
D000263:Adenylate Kinase / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010770:Phosphotransferases / Q000302:isolation & purification*; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D010770:Phosphotransferases; D000263:Adenylate Kinase; D002268:Carboxypeptidases; D010450:Endopeptidases; D014357:Trypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D003488:Cyanogen Bromide