Application of molecular modeling for prediction of substrate specificity in cytochrome P450 1A2 mutants.

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.

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

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/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)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.

The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism.

Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.