Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii.
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 Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.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 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.
Rao
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Radiation Biology & Biochemistry Division, Bhabha Atomic Research Centre, Mumbai, India. rbbd@magnum.barct1.ernet.in
D000596:Amino Acids / Q000032:analysis; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000254:growth & development; D008970:Molecular Weight; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D013762:Tetrahydrofolate Dehydrogenase