1. Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2
- Author
-
Byron Kemper, Balraj Doray, and Ci-Di Chen
- Subjects
Hemeprotein ,Recombinant Fusion Proteins ,Mutant ,Molecular Sequence Data ,Moths ,medicine.disease_cause ,Biochemistry ,Mixed Function Oxygenases ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,Catalytic Domain ,medicine ,Animals ,Amino Acid Sequence ,Sequence Deletion ,chemistry.chemical_classification ,Mutation ,COS cells ,biology ,Laurate hydroxylase activity ,Cytochrome P450 ,Molecular biology ,Peptide Fragments ,Amino acid ,Enzyme Activation ,chemistry ,Amino Acid Substitution ,Solubility ,COS Cells ,biology.protein ,Mutagenesis, Site-Directed ,Insect Proteins ,Cytochrome P-450 CYP4A ,Linker - Abstract
Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E. coli and insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.
- Published
- 1999