Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis.
In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors. Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant. Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase. The serine 31 mutation had little effect on the steady-state kinetic properties of the enzyme but produced a 100-fold increase in the dissociation constant (Kd) for the inhibitor methotrexate. The serine 34 mutant had much greater alterations in its properties than S31; specifically, S34 had a 3-fold reduction in the Km for NADPH, a 24-fold increase in the Km for H2folate, a 3-fold reduction in the overall reaction rate kcat, and an 80,000-fold increase in the Kd for methotrexate. In addition, the pH dependence of the steady-state kinetic parameters of S34 were different from that of the wild-type enzyme. These results suggest that phenylalanine 31 and phenylalanine 34 make very different contributions to ligand binding and catalysis in the human dihydrofolate reductase.
Schweitzer
B I
BI
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Srimatkandada
S
S
Gritsman
H
H
Sheridan
R
R
Venkataraghavan
R
R
Bertino
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase