Abstract
The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.
Highlights
A unique enzymatic reaction in which the synthesiosf adenosyltransferase) catalyzes an unusualenzymatic reaction the sulfonium center results fromdisplacement of the entire polyphosphate chain from MgATP
The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trappingmeasurements
The catalyticrate is strongly dependentonthe onine does not have a significant commitment to catal-concentration of K’, providing a convenient methodof alterysis at 0.25 mM KCl, intrinsicisotope effects ing turnover to aid in dtihaegnosis of intrinsic isotope effects
Summary
Decreasing the concentration ofKC1 decreased the reaction rate and caused a significant increase in the kinetic isotope effect (Table I1 and Fig. 2). At potassium concentrationsnear zero (no added KCl), the reaction rate was 60-fold lower than at saturating KC1 concentration, and the isotope effect increased to 1.128 & 0.003. A chase solution which contained a >50-fold molar excess of unlabeled substrate was added at 2 s, and over the approximately 60 s, aliquots were removed, mixed with an acid stop solution, and theamount of product was determined. In the experiment with ATP, the time course following chase addition extrapolated to a substantially higher amount of product formed in 2 s than is observed in the acid stop points. The increase in the amount of radioactive product present at 2 s in thechase experiment corresponds to 10% of the total amount of enzyme subunits present and gives a lower limit of0.10 for the fraction of enzyme-bound ATP which is converted to AdoMet in pkference to dissociation under these conditions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
