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Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli.

Kim DH, Kim KH, Isin EM, Guengerich FP, Chae HZ, Ahn T, Yun CH
Protein Expr Purif. 2008 57 (2): 188-200

PMID: 18032064 · DOI:10.1016/j.pep.2007.10.010

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.

MeSH Terms (19)

Amino Acid Sequence Base Sequence Codon Cytochrome P-450 CYP1A2 Cytochrome P-450 CYP1A2 Inhibitors Deuterium Enzyme Inhibitors Escherichia coli Humans Hydrogen Peroxide Hydroxylation Kinetics Molecular Sequence Data Mutation NADP Oxidation-Reduction Spectrophotometry Substrate Specificity Temperature

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