POINT MUTATIONS AT GLYCINE-121 OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE - IMPORTANT ROLES OF A FLEXIBLE LOOP IN THE STABILITY AND FUNCTION

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexibl e loop (residues 117-131), separated by 19 Angstrom from active site A sp27, was substituted by site-directed mutagenesis with eight amino ac ids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His), The free energy chan ge of unfolding decreased in the order of G121A>G121D>G121C>G121S, wil d-type>G121H>G121Y>G121L>G121V. The thermal denaturation temperature d ecreased with all mutations, accompanied by a decrease in the calorime tric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, K-m, was only slightly influenced, but k(cat) was significantly decreased by the mutations, there being 3- (G121C) to 4 2-fold (G121L) decreases in k(cat)/K-m compared to that of the wild-ty pe enzyme. The effects of mutations on the stability and enzyme activi ty were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activi ty with increases in the hydrophobicity and volume of amino acids sugg est 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.