Abstract
To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (_??_°) and adiabatic compressibility (_??_°) were determined for a series of mutants with amino acid replacements at Gly 67 (7 mutants), Gly 121 (6 mutants), and Ala 145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15°C. These mutations induced large changes in _??_° (0.710-0.733 cm3-g-1) and _??_° (-1.8×10-6-5.5×10-6 bar-1) from the corresponding values for the wild-type enzyme (_??_°=0.723 cm3.g-1, _??_s°=1.7×10-6 bar-1), probably due to modifications of internal cavities. The _??_s° value increased with increasing v°, but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between _??_s° and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large _??_s° value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (kcat) and in part due to increased affinity for the substrate (Km), despite the fact that the mutation sites are far from 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.
