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J. Biol. Chem., Vol. 278, Issue 6, 4021-4027, February 7, 2003

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Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6^*

Mark J. I. Paine^§, Lesley A. McLaughlin^§, Jack U. Flanagan, Carol A. Kemp^¶, Michael J. Sutcliffe^¶, Gordon C. K. Roberts, and C. Roland Wolf^**

From the  Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom the ^¶ Departments of Biochemistry and Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom, and the  Biological NMR Centre and Department of Biochemistry, University of Leicester, PO Box 138, University Road, Leicester LE1 9HN, United Kingdom

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K_m values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K_m 108 µM, k_cat 5 min¹) along with nifedipine (K_m 28 µM, k_cat 2 min¹⁾ and tolbutamide (K_m 315 µM, k_cat 1 min¹), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

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