Molecular basis for the differences in lidocaine binding and regioselectivity of oxidation by cytochromes P450 2B1 and 2B2

Biochemistry. 1998 Jan 6;37(1):311-8. doi: 10.1021/bi971528s.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Binding Sites / genetics
  • Chemical Fractionation
  • Cytochrome P-450 CYP2B1 / biosynthesis
  • Cytochrome P-450 CYP2B1 / genetics
  • Cytochrome P-450 CYP2B1 / metabolism*
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Kinetics
  • Lidocaine / metabolism*
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Rats
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Stereoisomerism
  • Steroid Hydroxylases / biosynthesis
  • Steroid Hydroxylases / genetics
  • Steroid Hydroxylases / metabolism*


  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Lidocaine
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP2B1
  • steroid 16-beta-hydroxylase