ABSTRACT
The relation between microenvironment and the tertiary structure of cytochrome P-450 LM2 has been investigated. No complete relaxation to the most active state of the native enzyme took place in the case of membrane-incorporated hemoprotein with three or four intramolecular cross-links. The spatial organization of the enzyme was predicted to determine the cross-link location on the hemoprotein surface and membrane-incorporated parts of the polypeptide chain. It was concluded on the basis of the predicted structure that hemoprotein has an amphipathic structure and, thus, the greater part of molecule is exposed to the water phase. Not more than one NH2-terminal alpha helix is able to incorporate into the membrane. The location of this region is believed to control the formation of the catalytically-active-conformational state of cytochrome P-450 LM2.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/metabolism , Animals , Binding Sites , Circular Dichroism , Cross-Linking Reagents/pharmacology , Imidoesters/pharmacology , Kinetics , Protein Conformation , Spectrophotometry , Substrate SpecificityABSTRACT
The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation. Whereas the complete inhibition of oxidation of type II substrate turnover needs two intramolecular cross-links, that of type I substrates necessitates at least seven cross-links. The incorporation of modified and native enzymes into the membrane lipid bilayer at temperatures above the phase transition point results in the enzyme activation. However, in case of the preimmobilized enzyme the activation does not reach the maximal values. Both stabilized and liposome-incorporated cytochrome P-450 can fully be reactivated via the cross-link disulfide bond reduction. No such effect is observed at temperatures below the phase transition point.
