Mechanisms of activation of phenacetin to reactive metabolites by cytochrome P-450: a theoretical study involving radical intermediates

The cytochrome P-450-mediated activation of phenacetin (PHEN) to reactive intermediates by two hypothetical mechanisms has been studied by use of SV 6-31G ab initio energy and spin distribution calculations. In our calculations, the cytochrome P-450 enzyme system has been substituted by a singlet oxygen atom in order to reduce the computational efforts and to fulfill the requirements as to spin conservation. Both mechanisms are based on the currently increasingly accepted view that radical intermediates, formed via sequential one-electron steps, play a crucial role in the metabolic activation of substrates by cytochrome P-450. The first pathway is proposed to involve an initial abstraction of an electron and a proton from the alpha-methylene carbon atom in the ethoxy side chain and can explain the O-deethylation products paracetamol and acetaldehyde. In the second pathway, an initial abstraction of an electron and a proton from the nitrogen atom in the acetylamino side chain is proposed. The calculated spin densities of the formed nitrogen radical indicate that the unpaired electron is primarily localized at the nitrogen atom and to a smaller extent at the ortho- and paracarbon atoms relative to the acetylamino group. Radical recombination reactions between a hydroxyl radical and the spin delocalization-radicalized reactive centers of the nitrogen radical can explain the formation of the metabolites N-hydroxy-PHEN, 2-hydroxy-PHEN, and the arylating metabolite N-acetyl-p-benzoquinone imine (NAPQI), which forms a 3-(S-glutathionyl)paracetamol conjugate in the presence of glutathione. NAPQI is proposed to be formed via intermediate formation of a hemiketal. Proposals are made for the decomposition of this hemiketal into NAPQI that are consistent with currently available experimental data on 14C- and 18O-labeled PHEN

A preliminary 3D model for cytochrome P450 2D6 constructed by homology model building

A homology model building study of cytochrome P450 2D6 has been carried out based on the crystal structure of cytochrome P450 101. The primary sequences of P450 101 and P450 2D6 were aligned by making use of an automated alignment procedure. This alignment was adjusted manually by matching alpha-helices (C, D, G, I, J, K and L) and beta-sheets (beta 3/beta 4) of P450 101 that are proposed to be conserved in membrane-bound P450s (Ouzounis and Melvin [Eur. J. Biochem., 198 (1991) 307]) to the corresponding regions in the primary amino acid sequence of P450 2D6. Furthermore, alpha-helices B, B' and F were found to be conserved in P450 2D6. No significant homology between the remaining regions of P450 101 and P450 2D6 could be found and these regions were therefore deleted. A 3D model of P450 2D6 was constructed by copying the coordinates of the residues from the crystal structure of P450 101 to the corresponding residues in P450 2D6. The regions without a significant homology with P450 101 were not incorporated into the model. After energy-minimization of the resulting 3D model of P450 2D6, possible active site residues were identified by fitting the substrates debrisoquine and dextrometorphan into the proposed active site. Both substrates could be positioned into a planar pocket near the heme region formed by residues Val370, Pro371, Leu372, Trp316, and part of the oxygen binding site of P450 2D6. Furthermore, the carboxylate group of either Asp100 or Asp301 was identified as a possible candidate for the proposed interaction with basic nitrogen atom(s) of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS

A homology model for rat mu class glutathione S-transferase 4-4

Glutathione S-transferases (GSTs) are an important class of phase II (de)toxifying enzymes, catalyzing the conjugation of glutathione (GSH) to electrophilic species. Recently, a number of cytosolic GSTs was crystallized. In the present study, molecular modeling techniques have been used to derive a three-dimensional homology model for rat GST 4-4 based upon the crystal structure of rat GST 3-3, both members of the mu class. GST 3-3 and GST 4-4 isoenzymes share a sequence homology of 88%. GST 4-4 distinguishes itself from GST 3-3 in being much more efficient and stereoselective in the nucleophilic addition of GSH to epoxides and alpha,beta-unsaturated ketones. GST 3-3, however, is much more efficient in catalyzing nucleophilic aromatic substitution reactions. In this study, several known substrates of GST 4-4 were selected and their GSH conjugates docked into the active site of GST 4-4. GSH conjugates of phenanthrene 9(S),10(R)-oxide and 4,5-diazaphenanthrene 9(S),10(R)-oxide were docked into the active site of both GST 3-3 and GST 4-4. From these homology modeling and docking data, the difference in stereoselectivity between GST 3-3 and GST 4-4 for the R- and S-configured carbons of the oxirane moiety could be rationalized. The data acquired from a recently derived small molecule model for GST 4-4 substrates were compared with the results of the present protein homology model of GST 4-4. The energy optimized positions of the conjugates in the protein model agreed very well with the original relative positions of the substrates within the substrate model, confirming the usefulness of small molecule models in the absence of structural protein data. The protein homology model, together with the substrate model, will be useful to further rationalize the substrate selectivity of GST 4-4, and to identify new potential GST 4-4 substrates

A theoretical study on the metabolic activation of paracetamol by cytochrome P-450: indications for a uniform oxidation mechanism

The cytochrome P-450 mediated activation of paracetamol (PAR) to the reactive electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI) has been studied by use of SV 6-31G ab initio energy calculations and spin distributions. A simplified model for cytochrome P-450 has been used by substituting the proposed biologically active ferric-oxene state of cytochrome P-450 by a singlet oxygen atom. The results indicate that an initial hydrogen abstraction from the phenolic hydroxyl group is favored by 30.1 kcal/mol over an initial hydrogen abstraction from the acetylamino nitrogen atom. Metabolic activation of PAR via primary formation of a phenoxy radical seems the most likely mechanism. The calculated ab initio spin densities indicate that the radical formed by hydrogen abstraction from the phenolic hydroxyl group stays predominantly localized at the phenolic oxygen. A second hydrogen abstraction from the acetylamino nitrogen atom, giving rise to the reactive intermediate NAPQI, is then favored in terms of energy differences. The unpaired electron of the phenoxy radical was found to delocalize only to a small extent toward the carbon atoms at the ortho and para positions relative to the hydroxyl-containing ring carbon, but nevertheless a recombination reaction between a hydroxyl radical and these radicalized carbon atoms at the ortho or para positions could explain the formation of the minor metabolites 3-hydroxy-PAR and p-benzoquinone plus acetamide

A predictive model for substrates of cytochrome P450-debrisoquine (2D6)

Molecular modeling techniques were used to derive a predictive model for substrates of cytochrome P450 2D6, an isozyme known to metabolize only compounds with one or more basic nitrogen atoms. Sixteen substrates, accounting for 23 metabolic reactions, with a distance of either 5 A ("5-A substrates", e.g., debrisoquine) or 7 A ("7-A substrates", e.g., dextromethorphan) between oxidation site and basic nitrogen atom were fitted into one model by postulating an interaction of the basic nitrogen atom with a negatively charged carboxylate group on the protein. This acidic residue anchors and neutralizes the positively charged basic nitrogen atom of the substrates. In case of "5-A substrates" this interaction probably occurs with the carboxylic oxygen atom nearest to the oxidation site, whereas in the case of "7-A substrates" this interaction takes place at the other oxygen atom. Furthermore, all substrates exhibit a coplanar conformation near the oxidation site and have negative molecular electrostatic potentials (MEPs) in a part of this planar domain approximately 3 A away from the oxidation site. No common features were found in the neighbourhood of the basic nitrogen atom of the substrates studied so that this region of the active site can accommodate a variety of N-substituents. Therefore, the substrate specificity of P450 2D6 most likely is determined by the distance between oxidation site and basic nitrogen atom, by steric constraints near the oxidation site, and by the degree of complementarity between the MEPs of substrate and protein in the planar region adjacent to the oxidation site.(ABSTRACT TRUNCATED AT 250 WORDS