title: Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase.
abstract: To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) 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(o) (0.710-0.733 cm(3). g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3). g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities. The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large beta(s)(o) 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 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.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan. gekko@sci.hiroshima-u.ac.jp.
authors.forename: K
authors.initials: K
authors.lastname: Gekko
authors.forename: T
authors.initials: T
authors.lastname: Kamiyama
authors.forename: E
authors.initials: E
authors.lastname: Ohmae
authors.forename: K
authors.initials: K
authors.lastname: Katayanagi
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D017354:Point Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase