A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.
Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.
Tsokos
D C
DC
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Omata
Y
Y
Robinson
R C
RC
Krutzsch
H C
HC
Gelboin
H V
HV
Friedman
F K
FK
D000595:Amino Acid Sequence; D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D016415:Sequence Alignment
D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System