Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2
Chen, Ci-Di
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https://hdl.handle.net/2142/87260
Description
Title
Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2
Author(s)
Chen, Ci-Di
Issue Date
1998
Doctoral Committee Chair(s)
Byron Kemper
Department of Study
Molecular and Integrative Physiology
Discipline
Molecular and Integrative Physiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.
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