title: A rapid screen of active site mutants in glycinamide ribonucleotide transformylase.
abstract: Specific and saturation site-directed mutageneses have been used to alter each polar residue within 6 A of the catalytic center of glycinamide ribonucleotide transformylase (EC 2.1.2.2). These mutants were rapidly screened for catalytic activity using functional complementation of auxotrophic cells. This screen allows a rapid qualitative estimate of enzyme activity for each of these mutants. These results have shown that none of the polar residues close to the catalytic center of the enzyme are irreplaceable, although several are important for full catalytic activity, namely, Asn106, His108, Ser135, and Asp144. A mechanism is proposed in which a fixed water molecule mediates the required proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleophilic attack by the amine of glycinamide ribonucleotide. The active site polar residues may act to alter the pKa values of the attacking and leaving amino groups within a putative tetrahedral intermediate in order to facilitate the transfer of the formyl group.
authors.affiliation: Department of Chemistry, Davey Laboratory, Pennsylvania State University, University park 16802, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Warren
authors.forename: A E
authors.initials: AE
authors.lastname: Marolewski
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005816:Genetic Complementation Test; D006860:Hydrogen Bonding; D019877:Hydroxymethyl and Formyl Transferases; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051026:Phosphoribosylglycinamide Formyltransferase; D015202:Protein Engineering; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
chemical_list: D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases