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S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.

Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important. The AdoMet synthetase mutants (i.e. MetK/C90A, MetK/C90S, and MetK/C240A) retained up to approximately 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity. Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90. Dimeric Cys-90 mutants retained approximately 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site. MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably all of the mutants are much more thermally stable than the wild type enzyme.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.