Protein control of the formation and decomposition of the CYP119 and CYP101 aryl-iron complexes |
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Authors: | R A Tschirret-Guth L S Koo PR Ortiz de Montellano |
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Institution: | (1) Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446, USA e-mail: ortiz@cgl.ucsf.edu Tel.: +1-415-4762903 Fax: +1-415-5024728, US |
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Abstract: | CYP119, the first thermophilic P450 enzyme, reacts much more slowly than CYP101 (P450cam) with aryldiazenes to give σ-bonded aryl-iron complexes. The CYP119 complexes are stable anaerobically at 80 °C but are readily oxidized by O2 to give the N-arylprotoporphyrin IX regioisomers. The aryl shift can also be initiated in the absence of O2 by K3Fe(CN)6. In contrast, the corresponding CYP101 complexes are insensitive to O2 but decompose at temperatures above 50 °C owing to denaturation of the protein. The rate of the CYP119 aryl shift is decreased
by electron-withdrawing substituents, with ρ=−1.50 for both the O2- and K3Fe(CN)6-dependent reactions. A similar dependence (ρ=−0.90) is observed for the K3Fe(CN)6-dependent CYP101 shift. The enthalpies and entropies of activation suggest that the CYP119 and CYP101 K3Fe(CN)6-mediated reactions are similar, but the CYP119 O2-dependent reaction proceeds via a different transition state. In all cases, the rate-determining step is oxidation of the
aryl-iron complex. The temperature dependence of the O2- and K3Fe(CN)6-dependent CYP119 shifts provides evidence for temperature-dependent equilibration of two active site conformations. The oxygen
sensitivity of the CYP119 aryl-iron complexes, and the temperature dependence of their rearragement, reflect the unique active
site properties of this thermophilic P450 enzyme.
Received: 12 August 1999 / Accepted: 8 December 1999 |
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Keywords: | CYP101 CYP119 Aryl-iron complexes Aryldiazenes Thermophilic P450 enzyme |
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