Evidence that the catalytic differences of two structurally homologous forms of cytochrome P-450 relate to their heme environment.
Cytochromes P-450 PB3a and PB3b, which appear to be equivalent to forms b and e described by Ryan et al. [Ryan, D.E., Thomas, P.E., & Levin, W. (1982) Arch. Biochem. Biophys. 216, 272-288], have been shown to share 97% sequence homology [Suwa, Y., Mizukami, Y., Sogawa, K., & Fujii-Kuriyama, Y. (1985) J. Biol. Chem. 260, 7980-7984] yet exhibit an intriguing difference in enzymatic activity. Studies to establish the basis for this difference, including a development of the technique of surface-enhanced resonance Raman spectroscopy (SERRS), are reported. Studies on substrate binding, metabolism, and redox properties, as well as SERRS, indicate a significant difference in the heme environment of these two proteins. No significant difference in the interaction of the two proteins with P-450 reductase could be established. However, this interaction appeared sensitive to changes in ionic strength, suggesting ionic interactions are important in the functional coupling of these electron-transport components. A marked variation in the ratio of PB3a to PB3b activity in the metabolism of different substrates, which included a series of structurally similar resorufin analogues, provided further evidence that reductase coupling was not a critical factor. Therefore, the few amino acid differences observed between these proteins indicate sites that may be important in influencing the heme environment of these cytochrome P-450's.
Wolf
C R
CR
Imperial Cancer Research Fund, University Department of Biochemistry, George Square, Edinburgh, U.K.
Miles
J S
JS
Seilman
S
S
Burke
M D
MD
Rospendowski
B N
BN
Kelly
K
K
Smith
W E
WE
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010449:Peptide Mapping; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D007527:Isoenzymes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital