ATP‐AMP phosphotransferase from Paracoccus denitrificans

Abstract

Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370-fold (overall yield of 44%, 554 U/mg, M , 22000, 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(ethy1ene glycol)-400. The enzyme is specific for adenine (deoxyadenine) nucleotides with K,,, values for ATP, ADP and AMP of 340 pm, 980 pM and 93 pM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 IM, 7.1 pM and 6.9 pM 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 "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. denirrificans 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 Ap,A to inhibit 95 % activity.Adenylate kinases are ubiquitous small monomeric intracellular enzymes ( M , 20000-32000) which catalyze the interconversion of adenine nucleotides according to the equation, MgATP + AMP + MgADP + ADP. The enzyme has been purified and characterized from a number of sources [l] and primary and tertiary structures of porcine and human adenylate kinases have been published [2-41. The catalytic mechanism of adenylate kinase and the detailed role(s) of this enzyme in vivo have not yet been elucidated. One approach to these problems is studying adenylate kinase (and functionally related enzymes) from different sources in order to take advantage of specific isozymes carrying special feature(s). In this paper we report the purification and characterization of adenylate kinase from Paracoccus denitrijkans and compare it with several other adenylate kinases. The microorganism is a simple bacterium having many mitochondria-like characteristics which are otherwise more randomly distributed among bacteria [5]. Adenylate kinase from P. denitrificans is of further interest because of its similarity with the enzyme from muscle (cytoplasm) rather than with the enzyme from mitochondria.Abbreviations. ApsA, adenosi...