S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization.
S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000201:enzymology; D008214:Lymphocytes / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000302:isolation & purification*; D008875:Middle Aged; D008970:Molecular Weight; D014166:Transferases / Q000302:isolation & purification*
D000596:Amino Acids; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D014166:Transferases; D008716:Methionine Adenosyltransferase; D008274:Magnesium