Abstract
Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.
Highlights
AdoMet synthesis takes place if adenyl-5’yl imidodiphosphate (AMP-PNP)is substituted for ATP
The Enzyme Preparation-A strain of E. coli which produces imidotriphosphate (PPNP) formed is not hydrolyzed, 80-fold more AdoMet synthetase was prepared by incorporatpermitting dissociation of AdoMet formation from tri- ing a plasmid containing the metK+ gene3 into a metJ host
Sodium dodecyl sulfate electrophoresis showed a single band of 43,000 molecular weight, indicating that thenative enzyme is a tetrameter of identical subunits
Summary
Hepes/(CH.r),N, pH 8.3, for 2 min and chromatographed on a column of Sephadex G-25 (0.5 X 20 cm), which was equilibrated and eluted with 25 mM Hepes/(CH&N, pH 8.3, containing 5 m~ MgClr and 0.5 mM '"Tl(acetate) (1700 cpm/nmol). With Mg'+ (which has a strong preference for coordination to oxygen rather than sulfulrigands) as the activating divalent metaiol n, AdoMet synthetase utilized the Aisomer of ATP@(as defined by Eckstein (22), S absolute configuration atthe&phosphoryl group (23)) as substrate. WhenMn2+ or Coz+, which coordinateeither oxygen or sulfur ligands, replacedMg2' as the activating divalent cation, AdoMet synthetase utilized both isomers of ATP@ as substrates. ~. bound to the @-phosphoryl group of ATPPS and indicates 4’,5’-Dehydroadenosineas a Possible Intermediate in the that on the enzyme Mg2+ coordinates to the pro-S oxygen AdoMet SynthetaseReaction-4’,5’-Dehydroadenosine,a nuatom on the@-phosphorylgroup of ATP (23). Positional interchange of oxygen atoms of the tripolyphosphate chainof ATP in the absenceof an overall reaction (24)
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