To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.
Keywords: 2-(N-morpholino)ethanesulfonic acid; 50mM MES, 25mM Tris 25mM ethanolamine (buffer); Active-site mutation; CD; CSM; Cavity and hydration; DHF; DHFR; Dihydrofolate reductase; Enzyme function; HSQC; High pressure; MES; MTE; MTE containing 100mM sodium chloride (buffer); MTEN; NMR; PEG; Pi; Solvent effect; THF; TMA; center of fluorescence spectral mass; circular dichroism; dihydrofolate; dihydrofolate reductase; heteronuclear single quantum correlation; nuclear magnetic resonance; phosphate; polyethylene glycol; tetrahydrofolate; tetramethylammonium.
© 2013.