Influence of Mutation of the Amino-Terminal Signal Anchor Sequence of Cytochrome P450 2B4 on the Enzyme Structure and Electron Transfer Processes.

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概要

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• The role of the NH2-terminal hydrophobic patch of cytochrome P4502B4 (CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) and cytochrome b5 (b5) was assessed using a variant lacking the signal anchor sequence (Δ2-27). CD, second-derivative, and fluorescence emission spectra indicated that the structure of the deletion mutant slightly differed from that of the native CYP2B4. Fitting of the initial-velocity patterns for P450R- and b5-directed electron transfer to the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an approximately 2.3-fold decrease in the affinity of the two electron donors for the engineered enzyme, while the reductive efficiency remained unaffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Δ2-27) accounted for this phenomenon. Interestingly, spectral docking of P450R to the truncated pigment was not hampered, while the binding of b5 was blocked. The rates of substrate-triggered aerobic NADPH consumption in systems containing CYP2B4(Δ2-27) and P450R were 16 to 56% those obtained with the unchanged hemoprotein. Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substrate-bound oxy complex. Experiments with b5 as the ultimate electron donor hinted at some damage to second-electron transfer to the truncated enzyme. The results are consistent with the proposal that the NH2-terminal hydrophobic region of CYP2B4 might be of importance in preservation of the catalytic competence of the enzyme.

• 社団法人 日本生化学会の論文

著者

• Lewis David

  Molecular Toxicology Group School Of Biological Sciences University Of Surrey.

• LEHNERER Michael

  Walther-Straub-Institut fur Pharmakologie und Toxikologie der LMU

• EULITZ Manfred

  Institut fur Klinische Molekularbiolgie und Tumorgenetik der GSF.

• HLAVICA Peter

  Walther-Straub-Institut fur Pharmakologie und Toxikologie der LMU.

• Pernecky Steven

  Department Of Chemistry Eastern Michigan University.

• Schulze Johannes

  Walther- Straub-Institut für Pharmakologie and Toxikologie der LMU

関連論文

• Identification of Key Residues in Rabbit Liver Microsomal Cytochrome P450 2B4:Importance in Interactions with NADPH-Cytochrome P450 Reductase^1
• Influence of Mutation of the Amino-Terminal Signal Anchor Sequence of Cytochrome P450 2B4 on the Enzyme Structure and Electron Transfer Processes
• Influence of Mutation of the Amino-Terminal Signal Anchor Sequence of Cytochrome P450 2B4 on the Enzyme Structure and Electron Transfer Processes.

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