The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies

Li Chung Hsu, Nai Chi Hsu, Julia A. Guzova, Victor M. Guzov, Shwu Fen Chang, Bon Chu Chung

Research output: Contribution to journalArticle

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Abstract

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169-173. Substitutions of Ile-171 and - 172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower V(max) value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.

Original languageEnglish
Pages (from-to)3306-3310
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number6
DOIs
Publication statusPublished - Feb 9 1996

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Steroid 21-Hydroxylase
Mutation
Kinetics
Proteins
Substitution reactions
Endopeptidase K
Mutagenesis
Apoproteins
Mutant Proteins
Amino Acid Substitution
Fractionation
Site-Directed Mutagenesis
Heme
Endoplasmic Reticulum
Fluorescent Antibody Technique
Conformations
Digestion
Catalyst activity
Membranes
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies. / Hsu, Li Chung; Hsu, Nai Chi; Guzova, Julia A.; Guzov, Victor M.; Chang, Shwu Fen; Chung, Bon Chu.

In: Journal of Biological Chemistry, Vol. 271, No. 6, 09.02.1996, p. 3306-3310.

Research output: Contribution to journalArticle

Hsu, Li Chung ; Hsu, Nai Chi ; Guzova, Julia A. ; Guzov, Victor M. ; Chang, Shwu Fen ; Chung, Bon Chu. / The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 6. pp. 3306-3310.
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AU - Chung, Bon Chu

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