Molecular basis for the differences in lidocaine binding and regioselectivity of oxidation by cytochromes P450 2B1 and 2B2.
The interactions of lidocaine (LIDO) with two closely related P450s, 2B1 and 2B2, were investigated using chimeric enzymes and single-point mutants derived from the two proteins. P450 2B1 exclusively catalyzed the N-deethylation of LIDO to generate monoethylglycinexylidide (MEGX) and 2B2 catalyzed both N-deethylation and hydroxylation reactions to generate MEGX and omega-diethylamino-2-hydroxymethyl-6-methylacetanilide. The addition of LIDO to 2B2 evoked a type I binding spectral change with a measured Ks of approximately 20 microM. The magnitude of the change in the absorbance obtained following the binding of LIDO to 2B2 was indicative of an approximately 30% switch of the heme iron to the high-spin form. In contrast, the addition of LIDO to 2B1 resulted in less than a 1% shift to the high-spin form even at LIDO concentrations as high as 10 mM. P450 2B2 exhibited a low Km value for LIDO (62 microM), whereas 2B1 had an approximately 10-fold higher Km value. However, the rates of LIDO oxidation by 2B1 were approximately 200-fold those exhibited by 2B2. Substitution of 2B2 residues by 2B1-derived amino acids influenced the spectral binding, regioselectivity of LIDO oxidation, and the kinetic properties of the enzyme. With the 2B2 Ala-363 to Val mutant, a complete switch of the 2B2 mutant to catalyzing only the N-deethylation activity was observed. The altered regioselectivity was accompanied with approximately 10-fold increases in the measured Ks, Km, and kcat values for LIDO.[1]References
- Molecular basis for the differences in lidocaine binding and regioselectivity of oxidation by cytochromes P450 2B1 and 2B2. Hanna, I.H., Roberts, E.S., Hollenberg, P.F. Biochemistry (1998) [Pubmed]
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