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 flexibilit y 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 a t Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measur ements at 15 degrees C. These mutations induced large changes in (v) over b ar(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 enzy me ((v) over bar(o) = 0.723 cm(3) . g(-1), <(beta)over bar>(o)(s) = 1.7 x 1 0(-6) bar(-1)), probably due to modifications of internal cavities, The <(b eta)over bar>(o)(s) value increased with increasing (v) over bar(o), but sh owed a decreasing tendency with the volume of the amino acid introduced. Th ere was no significant correlation between <(beta)over bar>(o)(s) and the o verall stability of the mutants determined from urea denaturation experimen ts. 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 influ enced by a single amino acid substitution in one of these loops and that th e flexible loops of this protein play important roles in determining the en zyme function.