The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2.
We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.
Narimatsu
Shizuo
S
Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. shizuo@pharm.okayama-u.ac.jp
Kiryu
Kimio
K
Yonemoto
Rei
R
Yoshino
Manabu
M
Kobatake
Mitsuko
M
Kazamori
Daichi
D
Hagino
Saori
S
Masuda
Kazufumi
K
Katsu
Takashi
T
Asanuma
Masato
M
Kumamoto
Takuya
T
Ishikawa
Tsutomu
T
Funae
Yoshihiko
Y
Yamano
Shigeru
S
Hanioka
Nobumitsu
N
Naito
Shinsaku
S
D008735:5-Methoxytryptamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D000072467:Cytochrome P450 Family 2; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D008861:Microsomes; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
C033696:5-methoxy-N,N-diisopropyltryptamine; D008735:5-Methoxytryptamine; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; C119985:Cyp2d2 protein, rat; D000072467:Cytochrome P450 Family 2