X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: Roles of Thr252 and water in the active center

Takako Hishiki, Hideo Shimada, Shingo Nagano, Tsuyoshi Egawa, Yasukazu Kanamori, Ryu Makino, Sam Yong Park, Shin ichi Adachi, Yoshitsugu Shiro, Yuzuru Ishimura

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H2O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

Original languageEnglish (US)
Pages (from-to)965-974
Number of pages10
JournalJournal of Biochemistry
Volume128
Issue number6
DOIs
StatePublished - Jan 1 2000

Keywords

  • Cytochrome P450cam
  • Oxygen activation
  • Proton transfer
  • Site-directed mutagenesis
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Hishiki, T., Shimada, H., Nagano, S., Egawa, T., Kanamori, Y., Makino, R., Park, S. Y., Adachi, S. I., Shiro, Y., & Ishimura, Y. (2000). X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: Roles of Thr252 and water in the active center. Journal of Biochemistry, 128(6), 965-974. https://doi.org/10.1093/oxfordjournals.jbchem.a022848