Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants.
Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5-7 angstroms from the site of the oxidation. The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, and p-biphenyldiazene, which react with P450 enzymes to form sigma-bonded aryl-iron (Fe-Ar) complexes. Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces the N-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, 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 pyrrole 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 determined by the same method for P450s for which crystal structures are available. Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in the N-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity. Replacement 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 abolishes catalytic activity. These results provide a refined model of the 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 ring B, but not to major structural reorganization of the active site.
Mackman
R
R
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Tschirret-Guth
R A
RA
Smith
G
G
Hayhurst
G P
GP
Ellis
S W
SW
Lennard
M S
MS
Tucker
G T
GT
Wolf
C R
CR
Ortiz de Montellano
P R
PR
D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D004563:Electrochemistry; D018698:Glutamic Acid; D005998:Glycine; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011524:Protoporphyrins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship
D006834:Hydrazines; D007097:Imines; D011524:Protoporphyrins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; C028025:protoporphyrin IX; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; D005998:Glycine