Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei: Effects of pH, salts, temperature, and source of NADPH on enzyme activity and substrate specificity studies

Abstract

Activity analyses of pure dihydrofolate reductase from amethopterin-resistant Lactobacillus casei conducted with commercial sources of NADPH yielded a progression of nonlinear assay tracings whose shapes were both pH dependent and reminiscent of classical product inhibition. The extent of curving of the assay tracings was dependent on the source and age of the commercial NADPH and was enhanced as the pH was decreased from 7.5 to 5.0. Under these conditions a “pseudo”-pH-activity profile, exhibiting a maximal specific activity of 9 units/mg of protein between pH 7.0 and 7.5, was found. In contrast, freshly prepared NADPH provided strictly linear assay tracings over the pH range of 8.5 to 5.0, yielding uniformly higher specific activities than those observed with commercial NADPH. The new pH-activity profile was characterized by a broad optimum between pH 5.0 and 6.0, with a maximal specificity activity of 24.9 units/ mg in 0.1 m potassium phosphate in the absence of added salt. The curving phenomenon and pseudo-pH optimum observed with commercial NADPH is attributed to the presence of minor but potent inhibitory impurities in these coenzyme preparations. Optimal concentrations of monovalent (~0.1 m) and divalent (~0.05 m) salts activated the enzyme between 1.5- and 1.7-fold, resulting in maximal specific activities in the range of 34 to 39 units/mg. A similar extent of activation was observed in 0.8 m Tris-acetate buffer, pH 5.5. At concentrations of monovalent salts above 0.5 m and of divalent salts above 0.2 m a reduction in salt-dependent activation and, in some cases, inhibition of activity were obtained. Substrate specificity studies indicated that the V for folate at saturating levels is 1% of that for dihydrofolate. Deamino-NADPH yielded V values 1.4-fold higher than that for NADPH, while acetylpyridine-NADPH and thio-NADPH provided values 6.5- and 235-fold lower, respectively, than the value with the natural coenzyme. Gel electrophoresis studies reflected a similar trend of selectivity in the interaction of NADPH and its analogs to form stable binary complexes. Stable ternary complexes of enzyme and amethopterin were formed with NADPH, deamino-NADPH, thio-NADPH, and acetylpyridine-NADPH. Although neither dihydrofolate nor NADP + and its analog form stable complexes with L. casei dihydrofolate reductase, both NADP + and deamino-NADP + interact with enzyme and dihydrofolate to generate stable ternary complexes.