Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli.
In this study, wild-type human CYP1A2 without the conventional N-terminal modification (second codon GCT) or the truncation of the N-terminal hydrophobic region was functionally expressed in Escherichia coli. Its enzymatic properties were compared with N-terminally modified CYP1A2. Although modified CYP1A2 is almost all high-spin, some wild-type CYP1A2 shifted to low-spin. Spectral binding titrations with several ligands could be performed with wild-type enzyme, but not with modified enzyme. Kinetic parameters for several substrates were similar for the two CYP1A2 enzymes. However, the oxidation rates of phenacetin by modified enzyme were approximately 2-fold higher than those by wild-type enzyme. The intermolecular isotope effects were approximately 2 for phenacetin O-deethylation catalyzed by both enzymes. However, the wild-type enzyme, but not the modified enzyme, increased C-hydroxylation when O-deethylation rates were lowered by deuterium substitution. Molecular switching indicates that phenacetin rotates within the active site of wild-type enzyme and suggests a looser conformation in the active site of the wild-type enzyme than of the modified enzyme. These results reveal that the overall enzymatic properties of wild-type CYP1A2 enzyme are quite similar to those of modified CYP1A2, although its active site environment seems to differ from that of the modified enzyme.
Kim
Dong-Hyun
DH
School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea.
Kim
Keon-Hee
KH
Isin
Emre M
EM
Guengerich
F Peter
FP
Chae
Ho Zoon
HZ
Ahn
Taeho
T
Yun
Chul-Ho
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D003903:Deuterium; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D013053:Spectrophotometry; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D003062:Codon; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004791:Enzyme Inhibitors; D009249:NADP; D003903:Deuterium; D006861:Hydrogen Peroxide; D019388:Cytochrome P-450 CYP1A2