Structural analysis of binding of a diamantoid substrate to cytochrome P450 2B4: possible role of Arg 133 in modulation of function and activity of this enzyme.
OBJECTIVE
Understanding the enzyme mechanism of P450 enzymes needs a detailed knowledge of substrate-enzyme interactions. Here, we examined the interaction of cytochrome P450 2B4 with a diamantanoid substrate.


METHODS
The interaction was followed using a photoactivable label, 3-azidiamantane. After photochemically driven reaction, the labeled enzyme was cleaved by trypsine and the labeled peptides separated by HPLC and identified with the help of radioactivity (for tritiated label) and mass spectrometry. The results were analysed on the basis of the known X-ray structures for mammalian cytochromes P450.


RESULTS
Identification of labeled peptides has shown that the probe (binding as a substrate to the enzyme) was attached to fragments: 30-48 (the most likely positions of the label are Leu44, Gln45 and Asp47), 127-140 (with Arg133 labeled, as indicated by mass spectrometry), 359-373 and 434-443 (the exact position of the label unknown). The structural comparison indicates considerable differences in Arg133 interaction with heme propionates, connected with binding of the substrate. Labeling of this residue may thus reflect its involvement in modulation of cytochrome P450 activity.


CONCLUSIONS
The results show existence of additional binding sites for substrate on cytochrome P450 2B4, located close to the surface of the enzyme.
Sulc
Miroslav
M
Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Hudecek
Jiri
J
Stiborova
Marie
M
Hodek
Petr
P
D000218:Adamantane / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics* / Q000502:physiology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D011492:Protein Hydrolysates / Q000737:chemistry; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
C121999:3-azidiamantane; D004791:Enzyme Inhibitors; D019920:Photoaffinity Labels; D011492:Protein Hydrolysates; D001120:Arginine; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D014357:Trypsin; D000218:Adamantane