Archaebacterial adenylate kinase from the thermoacidophile Sulfolobus acidocaldarius: purification, characterization, and partial sequence.
For the first time an adenylate kinase of archaebacterial origin has been purified to homogeneity. The enzyme from the extreme thermoacidophile Sulfolobus acidocaldarius (DSM 639) has been found to consist of a 23- to 24-kDa polypeptide likely to form a dimer under in vitro conditions. Its temperature optimum is about 90 degrees C; the pH optimum is 5.3-6.0, depending on the observed direction of the reaction. The KM values for ATP, AMP, and ADP are almost equal (0.6-0.7 mM). The enzyme is absolutely specific for AMP as phosphate acceptor but has a broad specificity for nucleotide triphosphates as donors. It requires divalent metals for activity with Mg2+ > Mn2+ > Ca2+ > Zn2+ in the order of decreasing potency. Distinct from mammalian enzymes the sensitivity toward the typical inhibitor diadenosine-5,5'-pentaphosphate is extremely low; > 300 microM is required for 50% inhibition, suggesting an altered distance between the AMP-/ATP-binding sites. The thermostability of the protein decreases sharply at room temperatures above 90 degrees C. It exhibits unusual stability, however, toward acidic pH. A 41-residue N-terminal sequence has been determined which allowed us to construct probes for the genetic approach. From residue 8-15 the protein contains the typical glycine rich P-loop as well as another conserved sequence stretch 21 residues further. Isolation of the gene is in progress.
Lacher
K
K
Institute of Biochemistry, Medical University of Lübeck, Germany.
Schäfer
G
G
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016945:Sulfolobus acidocaldarius / Q000201:enzymology* / Q000235:genetics
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase