title: Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid.
abstract: X-ray derived structural data predicted that serine 229 was positioned to act as a proton donor to the developing C2 carbanion during the reduction of enolpyruvyl-UDP-N-acetylglucosamine catalyzed by the bacterial peptidoglycan biosynthetic flavoenzyme MurB. To investigate this effect, a mutant where serine 229 was replaced by alanine was constructed and purified. Kinetic analysis of the two half-reactions for the mutant enzyme revealed a 9-fold decrease in the reduction of EFlox by NADPH and a dramatic 10(7)-fold decrease in the reoxidation of EFlred with the enolpyruvyl substrate. In addition, studies of S229A with the substrate analog, (E)-enolbutyryl-UDP-N-acetylglucosamine, showed a striking bias of the partitioning toward formation of the (Z) geometric isomer as opposed to formation of the reduced product UDP-methylmuramic acid, which was the predominant product in wild-type MurB. These studies provide evidence for the proposed role of this active-site serine as a general acid catalyst.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Benson
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: V
authors.initials: V
authors.lastname: Massey
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D011522:Protons; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D008024:Ligands; D011522:Protons; D012694:Serine; D009249:NADP; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase