The bifunctional active site of S-adenosylmethionine synthetase -: Roles of the active site aspartates

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP;) from ATP, and subsequently PPP, is hydrolyzed to PP, and Pi before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues, Aspartate residues Asp16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg2+ ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers, However, all have k(cat) reductions of similar to 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K-m values change by less than 3- and 8-fold, respectively, In the partial reaction of PPP, hydrolysis, mutants of the Mg2+ binding residues have >700-fold reduced catalytic efficiency (k(cat)/K-m), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP, hydrolysis by Mg2+ site mutants is improved by AdoMet, like the wild type enzyme, In contrast AdoMet reduces the catalytic efficiency for PPP, hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca2+ uniquely activates the D271A mutant enzyme to 15% of the level of Mg2+, in contrast to the similar to 1% Ca2+ activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca2+ and the smaller Mg2+.