Formation of a novel reversible cytochrome P450 spectral intermediate: role of threonine 303 in P450 2E1 inactivation.
tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds.
Blobaum
Anna L
AL
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
Lu
Yipin
Y
Kent
Ute M
UM
Wang
Shaomeng
S
Hollenberg
Paul F
PF
D000480:Alkynes / Q000494:pharmacology; D000693:Anaerobiosis; D000818:Animals; D001555:Benzene Derivatives / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004789:Enzyme Activation / Q000187:drug effects; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013057:Spectrum Analysis; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D000480:Alkynes; D001555:Benzene Derivatives; D065691:Cytochrome P-450 CYP2E1 Inhibitors; C489139:t-butyl acetylene; D013912:Threonine; D020122:tert-Butylhydroperoxide; D007501:Iron; D019392:Cytochrome P-450 CYP2E1; C007164:cumene hydroperoxide