title: The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues.
abstract: S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Taylor
authors.forename: G D
authors.initials: GD
authors.lastname: Markham
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011122:Polyphosphates / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary
chemical_list: D011122:Polyphosphates; D008716:Methionine Adenosyltransferase; C005692:triphosphoric acid