Purification and characterization of dihydrofolate reductase from <i>Lactobacillus leichmannii</i>

Abstract

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP⁺ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of <i>Lactobacillus leichmannii. </i>It has UV absorption maxima at 280 nm, <i>M</i>_r<i> </i>of 20,000, Stokes radius of 0.34 nm and a S_20,w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH₂- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC₅₀ = 2μ<i>M</i>). The enzyme has a <i>p</i>H optimum of 7.4 and is thermally inactivated at >35°C. It is activated by 0.1 <i>M </i>KCl and KI and 2<i>M </i>urea. 3-4<i>M </i>urea completely inactivated the enzyme. Enzyme has <i>K</i>_m<i> </i>values of 3.5 μ<i>M </i>and 6.2μ <i>M </i>for NADPH and DHF respectively, and a K_ivalue of 7 n<i>M</i> for MTX, the inhibition being competitive.