Purification and Properties of Dihydrofolate Reductase from Ehrlich Ascites Carcinoma Cells

J. R. Bertino, J. P. Perkins, D. G. Johns

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Abstract

The enzyme dihydrofolate reductase has been purified over 500-fold from Ehrlich ascites carcinoma cells. A molecular weight of 20,200 and a turnover number of 270 have been calculated. The Km values for reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and reduced nicotinamide-adenine dinucleotide are 5.6 × 10-6 and 2.6 × 10-4 m, respectively; oxidized nicotinamide-adenine dinucleotide phosphate has been found to be a competitive inhibitor (Ki = 4.9 × 10-6) of this enzyme reaction with respect to NADPH. The purified enzyme catalyzes the reduction of folate at a slower rate than the reduction of dihydrofolate, and only at an acid pH and with NADPH as the coenzyme. The ascites cell enzyme exhibited a broad specificity and reduced several pteroyl and dihydropteroyl compounds; N10-methyl-dihydrofolate, dichlorodihydrofolate, and dibromo-dihydrofolate were reduced at faster rates than was dihydrofolate. Studies of several inhibitors indicated that the 2,4-diamino structure resulted in enhanced binding of the inhibitor to the enzyme, and that other portions of the molecule, including the glutamic acid portion, the p-aminobenzoic acid ring, and in particular the N10-nitrogen are also important for binding. A Ki value of 6.1 × 10-9 for N10-formylfolate was calculated; this unusually low dissociation constant may explain the failure of this compound and its dihydro derivative to act as a substrate for dihydrofolate reductase.

Original languageEnglish (US)
Pages (from-to)839-846
Number of pages8
JournalBiochemistry
Volume4
Issue number5
DOIs
StatePublished - May 1 1965
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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