Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase.
Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme. Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme. The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme. Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme. Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme. The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar. The mutant enzymes were less thermostable than the wild-type enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.
Yubisui
T
T
Department of Biochemistry, Medical College of Oita, Japan.
Shirabe
K
K
Takeshita
M
M
Kobayashi
Y
Y
Fukumaki
Y
Y
Sakaki
Y
Y
Takano
T
T
D001483:Base Sequence; D001665:Binding Sites; D003579:Cytochrome Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D013816:Thermodynamics
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D009243:NAD; D012694:Serine; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase