Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli.
Protein Expr Purif
; 57(2): 188-200, 2008 Feb.
Article
en En
| MEDLINE
| ID: mdl-18032064
ABSTRACT
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Citocromo P-450 CYP1A2
/
Escherichia coli
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2008
Tipo del documento:
Article
Pais de publicación:
EEUU
/
ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA