To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His). The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V. The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme. The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction. Study holds ProTherm entries: 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919 Extra Details: additive : EDTA(0.1 mM), Activity at 25 degree C point mutations; glycine; Escherichia coli dihydrofolate reductase;,stability; free energy; flexible loop; function
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:20 p.m.
|Number of data points||98|
|Proteins||Dihydrofolate reductase ; Dihydrofolate reductase|
|Assays/Quantities/Protocols||Experimental Assay: activity_kcat ; Experimental Assay: Tm ; 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|