ATP-AMP phosphotransferase from Paracoccus denitrificans.
Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370-fold (overall yield of 44%, 554 U/mg, Mr 22 000, pI 4.7; pH optimum 7.5-8.0) by using successive column chromatography on DEAE-cellulose DE-52, Matrex-Blue A and Sephadex G-75. The enzyme was homogeneous by dodecylsulfate gel electrophoresis and constancy of specific activity across the single peak found in Sephadex G-75 chromatography. Crystals in the form of heavy needles have been obtained in poly(ethylene glycol)-400. The enzyme is specific for adenine (deoxyadenine) nucleotides with Km values for ATP, ADP and AMP of 340 microns, 980 microM and 93 microM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 microM, 7.1 microM and 6.9 microM respectively. The enzyme was noted to have no disulfide bond, two free sulfhydryl groups and a total of 208 residues. Aboe 50 mM KCl the enzyme activity drops off gradually. The enzyme is stable at neutral pH and below a temperature of 44 degrees C. Adenylate kinase from P. denitrificans resembles adenylate kinases from other sources with respect to such properties as the dependence on specific divalent cations, maximal activities at a ratio of Mg2+:ATP of about 1:1 and the ratio of Mg2+:ADP of 1:2, in having MgATP (or MgADP) binding site and another substrate binding site for AMP (or ADP), and in requiring an intact histidine and one or more arginine residues for enzymatic activity. Adenylate kinase from P. denitrificans appears to resemble the so-called muscle-type (cytoplasmic) rather than the mitochondrial-type adenylate kinase by comparisons of the following properties: low molecular weight, having --SH groups and no disulfide bonds, pH-optimum of 8.0 for formation of ADP, positive cross-reaction by ELISA test and in requiring a low concentration of Ap5A to inhibit 95% activity.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification*; D013379:Substrate Specificity
D000596:Amino Acids; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase