Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function.


Abstract

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

Submission Details

ID: aTfUsK3U4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Gekko K;Kunori Y;Takeuchi H;Ichihara S;Kodama M,J. Biochem. (1994) Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function. PMID:7798183
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