ACTIVE-SITE TOPOLOGIES OF HUMAN CYP2D6 AND ITS ASPARTATE-301-]GLUTAMATE, ASPARAGINE, AND GLYCINE MUTANTS

Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates wit h a positively charged nitrogen atom 5-7 Angstrom from the site of the oxidation. The active-site topology of CYP2D6 is examined here with p henyl,2-naphthhyl-, and p-biphenyldiazene, which react with P450 enzym es to form sigma-bonded aryl-iron (Fe-Ar) complexes. Ferricyanide-medi ated migration of the aryl group from the iron to the porphyrin nitrog ens produces the N-arylprotoporphyrin IX regioisomers (N-B:N-A:N-C:N-D , in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; and p-biphenyl, 16:84:00 :00. These results suggest that the CYP2D6 active site is open above p yrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D. This geometry differs from those determi ned by the same method for P450s for which crystal structures are avai lable. Replacement of Asp-301 by a Glu, which preserves the carboxylat e side chain, causes no detectable change in the N-aryl porphyrin regi oisomer patterns and only minor changes in the catalytic activity. Rep lacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually ab olishes catalytic activity. These results provide a refined model of t he active site of CYP2D6. They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ri ng B, but not to major structural reorganization of the active site. ( C) 1996 Academic Press, Inc.