Kinetics of oxidation of benzphetamine by compounds I of cytochrome P450 2B4 and its mutants

Abstract

Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized benzphetamine to norbenzphetamine in high yield in bulk studies. In direct kinetic studies of benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (K(bind)) for each. The first-order oxidation rate constants (k(ox)) were comparable for Compounds I of CYP2B4, the E301Q mutant, and the T302A mutant, whereas the k(ox) for Compound I of the F429H mutant was reduced by a factor of 2. CYP119 Compound I, studied for comparison purposes, reacted with benzphetamine with a binding constant that was nearly an order of magnitude smaller than that of CYP2B4 but a rate constant that was similar. Substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions, and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.

Figure 1

Stereo view of CYP2B4 active site showing the heme and positions of mutated residues (pdb:1SUO). Iron is the green ball in the center of heme, the negatively charged surfaces of E301 and T302 are shown in red, positively charged surfaces are in blue, and neutral surfaces are in white. The figure was generated with DS viewer Pro (Accelrys, Inc. San Diego CA).

Figure 2

Reactions for production of P450 Compound I via electron photo-ejection. The oval represents the porphyrin macrocycle in heme.

Figure 3

HPLC-mass spectrometry results. (A) Total ionization as a function of HPLC retention times. (B) Ionization in the range m/z 50,000 to 60,000 for the peaks eluting at 32 minutes, the maxima are at m/z = 55,723 (before PN) and m/z = 55,877 (after PN).

Figure 4

UV-visible spectrum of CYP2B4 Compound I. The LFP experiment gave an observed difference spectrum (dashed line) that was combined with a scaled spectrum of Compound II (dotted line) to give the Compound I spectrum (solid line).

Figure 5

Benzphetamine and its oxidation products hydroxybenzphentamine and norbenzphetamine.

Figure 6

Rate constants for reactions of Compounds I with benzphetamine: CYP2B4 (black), CYP2B4 E301Q (green), CYP2B4 T302A (red), CYP2B4 F429H (blue), and CYP119 (pink). The solid lines were generated from the kinetic parameters in Table 2.