EFFECT OF AMINO-ACID REPLACEMENT ON THE THERMAL TRANSITION AND STABILITY OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

Two mutant dihydrofolate reductases (DHFRs) were prepared by replacement of glycine-121 of the Eschrichia coli wild-type for alanine and valine by means of site-directed mutagenesis. Thermal denaturations (N-D transition) of the wild-type and the mutant DHFRs were investigated by high sensitivity differential scanning calorimetry. The enthalpy change associated with this transition increased in the order Val-DHFR<Ala-DHFR<wild(Gly)-DHFR, that is, in the decreasing order in the hydrophobicity of these residues at the position 121. The calorimetric data of these DHFRs were analyzed according to a double deconvolution method proposed by Kidokoro et al (Biopolymers, 26, 231 (1987)). Based on this method, the thermal transitions of three DHFRs were revealed to obey a three(N, I and D)-state model and the enthalpy functions of native, intermediate and denatared states, H(N)(T), H(I)(T) and H(D)(T), were calculated. The enthalpy difference between the native and intermediate states, DELTAH(NI) was shown to greatly differ among three DHFRs and increase in the same order as that of an increase in the N-D transition enthalpy. In accordance with this difference, the Gibbs free energy difference between the native and denatured states, DELTAG(ND), at temperatures below the phase transition also increased in the same order, indicating that the thermodynamic stability of DHFRs decreases with an increase in the hydrophobicity of amino acid residues at this position.