Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase

To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume ((v) over bar(o)) and adiabatic compressibility (<(beta)over bar>(o)(s)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C. These mutations induced large changes in (v) over bar(o) (0.710-0.733 cm(3) . g(-1)) and <(beta)over bar>(o)(s) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme ((v) over bar(o) = 0.723 cm(3) . g(-1), <(beta)over bar>(o)(s) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities, The <(beta)over bar>(o)(s) value increased with increasing (v) over bar(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between <(beta)over bar>(o)(s) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large <(beta)over bar>(o)(s) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K-m), despite the fact that the mutation sites are far hom the catalytic site,These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.