Abstract

Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of fourN-phenyl-protoporphyrin IX isomers (N_B:N_A:N_C:N_D, 01:01:02:02). In the presence of 10 mM MgCl₂, the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N_A isomer (N_B:N_A:N_C:N_D, 01:34:01:02). In addition, an investigation of MgCl₂effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl₂ increased the apparent K_mof triazolam 4-hydroxylation from 83 to 173 μM and reduced theV_max for the reaction from 3.4 to 2.4 min⁻¹. Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl₂, it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 ± 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl₂, the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 ± 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity.