Secobarbital-mediated inactivation of cytochrome P450 2B1 and its active site mutants. Partitioning between heme and protein alkylation and epoxidation.
Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.
He
K
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Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
He
Y A
YA
Szklarz
G D
GD
Halpert
J R
JR
Correia
M A
MA
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D004852:Epoxy Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D008297:Male; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012631:Secobarbital / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D004852:Epoxy Compounds; D012631:Secobarbital; D006418:Heme; D019362:Cytochrome P-450 CYP2B1