EFFECTS OF INSERTIONS AND DELETIONS IN A BETA-BULGE REGION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

The role of a beta-bulge in Escherichia coli dihydrofolate reductase ( DHFR) has been explored by a series of insertion and deletion mutation s, Insertion of a seven amino acid sequence from a structurally equiva lent 'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (k(cat)K(m)) 34-fo ld. Deletion of F137, Delta 137, the looped out residue in the bulge, also destabilizes E. coli DHFR by 2.8 kcal/mol but only decreases cata lytic efficiency threefold. Concurrent deletion of F137 and mutation o f V136 to proline to try and maintain the strand twist associated with the beta-bulge decreases protein stability by 3.4 kcal/mol and decrea ses catalytic efficiency 84-fold. These insertion/deletion mutations w ere also constructed in a D27S DHFR background, The D27S mutation has been described previously and proposed to remove the catalytic acid fr om the active site, The Delta 137 mutation partially suppresses the ef fect of the D27S mutation as it decreases the K-m for substrate, dihyd rofolate, twofold. Non-additive effects are observed for the insertion /deletion mutations in wild-type versus D27S DHFR backgrounds, consist ent with structural changes.