Investigation of Monovalent Cation Activation of S-Adenosylmethionine Synthetase Using Mutagenesis and Uranyl Inhibition

摘要: S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet from ATP and L-methionine with subsequent hydrolysis bound tripolyphosphate intermediate. Maximal activity requires presence two divalent one monovalent cation per active site. Recently, x-ray structure Escherichia coli was solved, positions Mg2+ binding sites were identified. Based on additional spherical electron density, K+ site postulated to be a nearby where uranyl heavy atom derivative also in crystal. The side chain glutamate 42 is within ligation distance metals. Mutagenesis glutamine (E42QMetK) abolished activation produced an enzyme that has kinetic properties virtually identical those K(+)-free wild type both overall reaction tripolyphosphate. Thus, there approximately 100-fold decrease Vmax for synthesis large increases Km values substrates. In contrast only 2-fold hydrolysis. ion, UO2(2+), competitive inhibitor respect (Ki = 350 nM) first ion bind at this inhibit enzyme. UO2(2+) inhibition reversible tight-binding, results not UO2(2+)-ATP. Analogous activation, predominantly inhibits rather than indicate likely result interference productive binding. remains tight-binding E42Q mutant, which suggests have different preferences when pocket. support model provides ligands major role