期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 436, 期 1, 页码 40-49出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2004.12.026
关键词
cytochrome P450-CAM; oxyferrous cytochrome P450-CAM; substrate-free oxy-cytochrome P450-CAM; magnetic dichroism; camphor analogs; T252A cytochrome P450-CAM mutant; 5-methylenylcamphor
资金
- NIGMS NIH HHS [GM 26730, GM 31756] Funding Source: Medline
To probe whether the nature of the Substrate can directly influence the spectral properties of oxyferrous cytochrorne P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at similar to 355, similar to 420, similar to 554, and similar to 585 nm (shoulder) and a characteristic MCD trough at similar to 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation Substrate) appears to have an anomalous binding mode for the Mutant compared with that for the wild-type enzyme. The present results indicate that the Structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme. (c) 2005 Elsevier Inc. All rights reserved.
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