Reversible Pressure Deformation of A Thermophilic Cytochrome P450 Enzyme (CYP119) and Its Active-Site Mutants
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- 作者：Richard A. Tschirret-Guth ; Laura S. Koo ; Gaston Hui Bon Hoa ; and Paul R. Ortiz de Montellano
- 刊名：Journal of the American Chemical Society
- 出版年：2001
- 出版时间：April 18, 2001
- 年：2001
- 卷：123
- 期：15
- 页码：3412 - 3417
- 全文大小：90K
- 年卷期：v.123,no.15(April 18, 2001)
- ISSN：1520-5126
文摘
The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-siteThr213 and Thr214 mutants was investigated. At 20 
C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volumeof activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolutereaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decompositionof iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variationof the N-arylprotoporphyrin-IX regioisomer (NB:NA:NC:ND) adduct pattern from 39:47:07:07 at 0.1 MPa to23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation anddecomposition gave the same regioisomer distribution as untreated protein. The results indicate that the proteinis reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450cam and other P450enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.
C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volumeof activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolutereaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decompositionof iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variationof the N-arylprotoporphyrin-IX regioisomer (NB:NA:NC:ND) adduct pattern from 39:47:07:07 at 0.1 MPa to23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation anddecomposition gave the same regioisomer distribution as untreated protein. The results indicate that the proteinis reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450cam and other P450enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.