Abstract
Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35% decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33% that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38% wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild-type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis.
Highlights
Substrate and inhibitor binding to dihydrofolarete- Dihydrofolate reductase (DHFR)’ (tetrahydrofolate: ductase (DHFR) primarily involvesresiduesin the NADP+oxidoreductase, EC 1.5.1.3.), which catalyzes the nicamino-terminal half of the enzyme; antibody otinamide adenine dinucleotide phosphate binding studies performed in this laboratory suggest(eNdADPH)-dependent reduction of 7,8-dihydrofolate (FAH,)
That the loop region located in the carboxyl terminus to tetrahydrofolate (FAH4),is necessary for maintaining inof human DHFR is in- tracellular pools of tetrahydrofolate and itsderivatives, which volved in conformational changes that occur upon li- provide single-carbon groups for many biosynthetic reactions
Vertebrate enzymes are about 30 amino acid residues larger the del-4 mutant enzyme retained a specific activity than thebacterial enzymes, and most of these extra residues almost 33% that of the wild type
Summary
DNAs-The pDFR expression system for hDHFR has been described previously (Prendergastet al., 1988). Plasmid DNA was isolated by the alkaline lysis method (Maniatis, 1982) followedby passage through a pZ523 column (5Prime-3Prime) and subsequent RNase treatment. The construction of the single-stranded template used for site-directedmutagenesis is described in detail elsewhere (Thompson and Freisheim, 1991). The cDNA encoding all but the first 22 amino acids was cloned into M13, and thesingle-stranded form of this recombinant phage was used as the template. The 18base oligodeoxynucleotides used for mutagenesis, 5"CCAGGTGTT-. GAG-3'(del-4), and 5'-GAATACCCACAGGAGGAG-3'(del-6),as well as three primers used in sequencing were synthesized in this laboratoryusing an Applied Biosystems model 380A instrument
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