To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction. Study holds ProTherm entries: 2896, 2897, 2898, 2899, 2900, 2901 Extra Details: additive : EDTA(0.1 mM), point mutations; glycine; Escherichia coli dihydrofolate reductase;,stability; free energy; flexible loop
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:20 p.m.
|Number of data points||35|
|Proteins||Dihydrofolate reductase ; Dihydrofolate reductase|
|Assays/Quantities/Protocols||Experimental Assay: activity_kcat ; Experimental Assay: Tm ; Experimental Assay: dHcal ; Experimental Assay: dHvH ; Experimental Assay: activity_km ; Experimental Assay: Cm ; Experimental Assay: activity_kcat temp:15.0 C ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: activity_km temp:15.0 C ; Derived Quantity: dTm ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR|