Alteration in catalytic properties of human CYP2D6 caused by substitution of glycine-42 with arginine, lysine and glutamic acid.
The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.
Tsuzuki
Daisuke
D
Laboratories of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Hichiya
Hiroyuki
H
Okuda
Yukie
Y
Yamamoto
Shigeo
S
Tamagake
Keietsu
K
Shinoda
Sumio
S
Narimatsu
Shizuo
S