The cytochrome P450 1A2 active site: topology and perturbations caused by glutamic acid-318 and threonine-319 mutations.
Phenyldiazene reacts with rat liver CYP1A2 expressed in Saccharomyces cerevisiae to give a phenyl-iron complex that rearranges to a mixture (NB:NA:NC:ND = 12:54:14:20, subscript indicates pyrrole ring) of N-phenyl-PPIX (PPIX = protoporphyrin) regioisomers. The same isomer pattern is obtained in each instance when the purified or microsomal enzyme reacts with phenyldiazene, indicating that the active site topology is not altered by removal of the protein from the membrane. Reaction of the enzyme with biphenylhydrazine gives a similar distribution of N-biphenyl-PPIX isomers, but reaction with (2-naphthyl)-hydrazine only gives the NC and ND regioisomers and a trace of the NA isomer of N-(2-naphthyl)-PPIX. The mutations E318D, E318A, and E318V cause relatively minor changes in the observed regioisomer ratios. In contrast, the mutations T319A, T319V, and T319S suppress formation of the NC and ND isomers of N-phenyl-PPIX. The reaction of T319A with biphenylhydrazine yields major amounts of the NB adduct rather than the small amounts observed with CYP1A2 and the Glu-318 mutants, but does not give the NC and ND regioisomers. Other, less dramatic, changes in the isomer ratios are also observed. The results indicate that the active site of CYP1A2 is open above all four quadrants of the heme group including, to some extent, the region above pyrrole ring B. Pyrrole ring B is completely inaccessible in most cytochrome P450 enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Tuck
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Hiroya
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Shimizu
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Hatano
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Ortiz de Montellano
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PR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D007097:Imines; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013912:Threonine
D005971:Glutamates; D007097:Imines; D011994:Recombinant Proteins; D013912:Threonine; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2