Role of Lys-110 of human NADH-cytochrome b5 reductase in NADH binding as probed by site-directed mutagenesis.
Lys-110 of human NADH-cytochrome b5 reductase was replaced by Ala, Met, or Arg by site-directed mutagenesis to evaluate the role of the residue. Km values of purified Lys-110-->Ala and Lys-110-->Met mutants for NADH were approximately 200-fold and 1,100-fold higher than that of the wild-type, respectively, while the value of the Arg mutant was almost the same as that of the wild-type. These results indicate that the positive charge at position 110 is important for NADH binding. The kcat value of Lys-110-->Ala was not affected, indicating that the residue only participates in the binding process in the reaction by forming an ionic interaction with phosphoryl group of NADH.
Fujimoto
Y
Y
Department of Biochemistry, Oita Medical University, Japan.
Shirabe
K
K
Nagai
T
T
Yubisui
T
T
Takeshita
M
M
D001483:Base Sequence; D003579:Cytochrome Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D004277:DNA, Single-Stranded; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009243:NAD; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D008239:Lysine