title: Molecular basis for the differences in lidocaine binding and regioselectivity of oxidation by cytochromes P450 2B1 and 2B2.
abstract: 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.
authors.affiliation: Department of Pharmacology, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: I H
authors.initials: IH
authors.lastname: Hanna
authors.forename: E S
authors.initials: ES
authors.lastname: Roberts
authors.forename: P F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites / Q000235:genetics; D005591:Chemical Fractionation; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008012:Lidocaine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013237:Stereoisomerism; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D008012:Lidocaine; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C025619:steroid 16-beta-hydroxylase