Homology modelling of human CYP2E1 based on the CYP2C5 crystal structure: investigation of enzyme–substrate and enzyme–inhibitor interactions

Abstract

The construction of a homology model of human cytochrome P450 2E1 (CYP2E1) is reported, based on the CYP2C5 crystallographic template. A relatively high degree of primary sequence homology (identity=59%), as expected for proteins of the same CYP family, ensured a straightforward generation of the 3-dimensional model due to relatively few deletions and insertions of amino acid residues with respect to the CYP2C5 crystal structure. Probing the CYP2E1 model with typical substrates of the enzyme showed a good agreement with experimental information in the form of positions of metabolism for substrates, and with site-directed mutagenesis data on certain residues. Furthermore, quantitative relationships between substrate binding affinity and various structural parameters associated with the substrate molecules facilitated the formulation of a procedure for estimating relative binding energy and, consequently, K_m or K_D values towards the CYP2E1 enzyme. This method has been based on a consideration of the active site interactions between substrates and key amino acid residues lining the haem pocket, together with compound lipophilicity data from partition coefficients.

Introduction

The cytochromes P450 (CYP) constitute a superfamily of heme-thiolate enzymes, of which over 2700 individual members are currently known, and are present in most species for the Phase 1 metabolism of drugs and other xenobiotics, although endogenous roles have also been characterized (Gonzalez, 1992, Wrighton and Stevens, 1992, Lewis, 1996, Lewis, 2001, Parkinson, 1996, Anzenbacher and Anzenbacherova, 2001, Guengerich, 2002). Enzymes of the CYP1, CYP2 and CYP3 families represent P450s most closely associated with the metabolism of foreign compounds in mammalian species, with the CYP2 family as a whole being involved in the greater part of P450-mediated drug oxidations in man (Rendic and DiCarlo, 1997, Evans and Relling, 1999). Although the CYP2E1 enzyme only plays a relatively minor role (Rendic and DiCarlo, 1997) in human drug metabolism (approx. 4% involvement in total drug oxidations known to be mediated by P450s), it is apparent that a number of low molecular weight carcinogens and other toxicants undergo metabolic transformations via CYP2E-mediated pathways (Guengerich et al., 1991, Liu et al., 1993, Raucy et al., 1993, Terelius et al., 1993, Gonzalez and Gelboin, 1994, Kukielka and Cederbaum, 1994, Cai and Guengerich, 2001). Table 1 summarizes the metabolic properties of a number of typical human CYP2E1 substrates, including drugs, solvents and other industrial chemicals (Ronis et al., 1996, Rendic and DiCarlo, 1997). There is a relatively high degree of homology between human CYP2E1 and orthologous proteins from other mammalian species, such as the mouse and rat, although some species differences in CYP2E1-mediated metabolism exist such as that shown by butadiene (Melnick and Kohn, 1995, Lewis et al., 1997, Lewis et al., 2000, Bird et al., 2001). In addition, it has been reported that a small number of allelic variants of human CYP2E1 exist, although these appear to exhibit only minor alteration in catalytic activity relative to the wild-type (Ingelman-Sundberg, 2001). Some of the physicochemical properties of CYP2E1 substrates are presented in Table 2, from which it can be appreciated that the majority are neutral, small molecular weight compounds with relatively low log P values (where P is the octanol/water partition coefficient). Apart from their relatively small molecular size being a common factor, CYP2E1 substrates exhibit structural diversity, although some contain a single aromatic ring and this is also a feature shown by certain inhibitors of the enzyme (Hargreaves et al., 1994, Rodrigues, 1999), with 3-amino-1,2,4-triazole representing a selective inhibitor of human CYP2E1 (Koop, 1990). The availability of the crystallographic co-ordinates for the rabbit enzyme CYP2C5, a mammalian enzyme from within the same family, has provided an opportunity for developing homology models of CYP2 enzymes (Lewis, 2002) and there is an interest in human CYP2E1 from the point of view of evaluating the likely metabolic fate of small molecular weight environmental agents in Homo sapiens. CYP2C5 exhibits significantly high primary sequence identity with CYP2E1 (59%) than that shown by the previously used template CYP102 (25%) in our earlier studies (Lewis et al., 1997, Lewis et al., 2000). Consequently, we have utilized the CYP2C5 crystal structure as a template for modelling human CYP2E1 such that the interactions of typical substrates and inhibitors can be investigated.