Key residues controlling phenacetin metabolism by human cytochrome P450 2A enzymes

Abstract

Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.

Figure 1

Spectral changes upon phenacetin binding to (A) CYP2A13, (B) CYP2A6, (C) CYP2A13 A301G, and (D) CYP2A6 I208S/I300F/G301A/S369G. Increasing concentrations of phenacetin during the titrations are indicated by spectra scans colored from red (low concentration 0.5 μM) to indigo (high concentration 250–300 μM).

Figure 2

Effects of mutation on phenacetin O-deethylation rates by CYP2A13 mutant proteins.

Figure 3

Effects of mutation on phenacetin O-deethylation rates by CYP2A6 mutant proteins.

Figure 4

Phenacetin metabolism by CYP1A2, CYP2A13, CYP2A13 and CYP2A6 mutant proteins. Data represents a global fit to two independent experiments with each point performed in triplicate.

Figure 5

The CYP2A6 I208S/I300F/G301A/G369S structure. A. Electron density surrounding the phenacetin ligand found in molecule B of the CYP2A6 I208S/I300F/G301A/G369S structure. B. Structural overlay of the four non-identical molecules found in the CYP2A6 I208S/I300F/G301A/G369S asymmetric unit, highlighting the differences in phenacetin and L370 positions. Molecules A (green), B (yellow), and D (blue) contain one molecule of phenacetin, while molecule C (pink) did not have electron density for phenacetin. D. Active site volume comparison between molecule A (green mesh, 300 ų) and molecule B (yellow mesh, 270 ų). The difference in active site volume among the four CYP2A6 quadruply-mutated protein molecules appears to depend largely on the position of L370. All structure figures were made with Pymol (DeLano, 2002). Hydrogen bonding to N297 is indicated by black dashed lines.

Figure 6

Comparison of the active sites of CYP2A6 (purple, PDB 1Z10, molecule B), CYP2A6 I208S/I300F/G301A/G369S with phenacetin (green, molecule A), and CYP2A13 (orange, PDB 2P85, molecule A). The four residues mutated in the CYP2A6 quadruple mutant, identical to the residues found in CYP2A13, are labeled in bold. The hydrogen bond from phenacetin to N297 is shown as a dashed black line.

Figure 7

Stereo view comparison of the active site cavities of CYP2A6 (purple protein and mesh, PDB 1Z10), CYP2A13 (orange protein and mesh, PDB 2P85), and CYP2A6 I208S/I300F/G301A/G369S (green protein and mesh). Steric clashes between the phenacetin and CYP2A6 residues 300, 209, and 370 are evident.