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ABSTRACT:Molecular dynamics (MD) simulations of 7-ethoxy-and 7-methoxyresorufin bound in the active site of cytochrome P450 (P450) 1A2 wild-type and various mutants were used to predict changes in substrate specificity of the mutants. A total of 26 multiple mutants representing all possible combinations of five key amino acid residues, which are different between P450 1A1 and 1A2, were examined. The resorufin substrates were docked in the active site of each enzyme in the productive binding orientation, and MD simulations were performed on the enzyme-substrate complex. Ensembles collected from MD trajectories were then scored on the basis of geometric parameters relating substrate position with respect to the activated oxoheme cofactor. The results showed a high correlation between the previous experimental data on P450 1A2 wild-type and single mutants with respect to the ratio between 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) activities and the equivalent in silico "E/M scores" (the ratio of hits obtained with 7-ethoxyresorufin to those obtained with 7-methoxyresorufin). Moreover, this correlation served to establish linear regression models used to evaluate E/M scores of multiple P450 1A2 mutants. Seven mutants, all of them incorporating the L382V substitution, were predicted to shift specificity to that of P450 1A1. The predictions were then verified experimentally. The appropriate P450 1A2 multiple mutants were constructed by site-directed mutagenesis, expressed in Escherichia coli, and assayed for EROD and MROD activities. Of six mutants, five demonstrated an increased EROD/MROD ratio, confirming modeling predictions.Cytochromes P450 constitute a large family of heme-thiolate monooxygenase enzymes widely found in nature (Nelson et al., 1996). These enzymes are capable of catalyzing the oxidation of a wide variety of both xenobiotic and endogenous compounds. However, even closely related isoforms may exhibit different catalytic activities.Human P450 1A1 and 1A2, the two major isoforms in the P450 1A subfamily, share 72% amino acid sequence identity but display different substrate specificities. P450 1A1 prefers to metabolize benzo- [a]pyrene and other polycyclic aromatic hydrocarbons, whereas P450 1A2 favors the oxidation of heterocyclic and aromatic amines (Kawajiri and Hayashi, 1996;Guengerich, 2005). Likewise, they also exhibit different substrate specificities with resorufin substrates such as 7-ethoxyresorufin and 7-methoxyresorufin (Nerurkar et al., 1993;Burke et al., 1994). Therefore, the P450 1A1/1A2 system provides a good model for exploring the basis for functional differences between individual P450 enzymes.The experimentally determined structure of a protein can provide valuable insight into its function. Homology modeling is an alternative method for obtaining the structure when the crystal structure is not available (Szklarz et al., 2000). Molecular dynamics (MD) simulations on the enzyme-substrate complex could provide information on whether the substrat...

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H 2 O 2 or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/ ⅐ SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine ؉ HO ⅐ ) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/ ⅐ SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically 13 C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H 2 O 2 solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.