Conversion of arginine to lysine at position 70 of human dihydrofolate reductase: generation of a methotrexate-insensitive mutant enzyme.
Arginine-70 of human dihydrofolate reductase (hDHFR) is a highly conserved residue which X-ray crystallographic data have shown to interact with the alpha-carboxylate of the terminal L-glutamate moiety of either folic acid or methotrexate (MTX). The rationale for this study was to introduce a conservative amino acid residue change at position 70 (Arg----Lys) which might function as a titratable group and, thus, reveal possible quantitative changes in ligand binding and kinetic parameters as a function of pH. Such a mutant enzyme (R70K) has been constructed and expressed by using site-directed mutagenesis techniques. This substitution has a dramatic effect on the binding of MTX, which displays a 22,600-fold increase in the dissociation constant (KD) at pH 7.5 compared to that of the reported wild-type enzyme value. At this pH, the KD value for dihydrofolate (FAH2) for the R70K enzyme shows only a 7-fold increase over that for the wild-type hDHFR. The pH profiles of the Michaelis and dissociation constants for FAH2 and KD values for MTX for the mutant enzyme all show a 7-8-fold increase from pH 7.5 to 8.5 as compared to its wild-type counterpart. The binding of NADPH or the nonclassical inhibitor trimetrexate (TMQ) to either the wild-type or the mutant enzyme does not show such pH-dependent characteristics. Thus, since FAH2 and MTX interact with the guanidinium side chain of arginine-70 in the wild-type hDHFR, the replacement of this residue with a lysine in the R70K mutant appears to have resulted in the introduction of a titratable group with a perturbed pKa value of ca. 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)
Thompson
P D
PD
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699.
Freisheim
J H
JH
D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D005493:Folic Acid Antagonists; D005785:Gene Conversion; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D005493:Folic Acid Antagonists; D001120:Arginine; D013762:Tetrahydrofolate Dehydrogenase; D008239:Lysine; D008727:Methotrexate