Functional role of residues in the helix B′ region of cytochrome P450 2B1

• Published in: Archives of biochemistry and biophysics Vol. 435; no. 1; pp. 157 - 165
• Main Authors: Honma, Wataru, Li, Weihua, Liu, Hong, Scott, Emily E., Halpert, James R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2005
• Subjects: Amino Acid Substitution; Amino Acids - chemistry; Amino Acids - genetics; Amino Acids - metabolism; Binding Sites; Cytochrome P-450 CYP2B1 - chemistry; Cytochrome P-450 CYP2B1 - genetics; Cytochrome P-450 CYP2B1 - metabolism; Cytochrome P450; Models, Chemical; Models, Molecular; Monooxygenase; Protein Binding; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins - chemistry; Site-directed mutagenesis; Structure-Activity Relationship; Testosterone - chemistry; Testosterone - metabolism; Monooxygenase; Cytochrome P450; Site-directed mutagenesis
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2004.12.014

Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B′ when ligands bind. To investigate the functional role of helix B′ in P450 2B1, residues 100–109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresorufin, 7-ethoxy-4-trifluoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in k cat values and significant differences in catalytic efficiencies among the substrates examined, consistent with structural information suggesting that the helix B′ region can adopt multiple conformations with different contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The findings suggest that residues in the helix B′ region affect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry.