Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase.
Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of Km values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating Km and Kcat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The Km for NADPH for the K54Q mutant enzyme is 58-fold higher, while the Km for NADH for K54Q is only 3.9-fold higher than that of the wild type, indicating that the substitution of Lys-54 with Gln-54 decreases the apparent affinity of the enzyme for NADPH dramatically, but has a lesser effect on the apparent affinity for NADH.(ABSTRACT TRUNCATED AT 250 WORDS)
Huang
S
S
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008.
Appleman
R
R
Tan
X H
XH
Thompson
P D
PD
Blakley
R L
RL
Sheridan
R P
RP
Venkataraghavan
R
R
Freisheim
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D008239:Lysine