A RAPID SCREEN OF ACTIVE-SITE MUTANTS IN GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE

Specific and saturation site-directed mutageneses have been used to al ter each polar residue within 6 Angstrom of the catalytic center of gl ycinamide ribonucleotide transformylase (EC 2.1.2.2). These mutants we re rapidly screened for catalytic activity using functional complement ation of auxotrophic cells. This screen allows a rapid qualitative est imate of enzyme activity for each of these mutants. These results have shown that none of the polar residues close to the catalytic center o f the enzyme are irreplaceable, although several are important for ful l catalytic activity, namely, Asn106, His108, Ser135, and Asp144. A me chanism is proposed in which a fixed water molecule mediates the requi red proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleoph ilic attack by the amine of glycinamide ribonucleotide. The active sit e polar residues may act to alter the pK(a) values of the attacking an d leaving amino groups within a putative tetrahedral intermediate in o rder to facilitate the transfer of the formyl group.