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J Biol Chem. 2003 Feb 7;278(6):4021-7. Epub 2002 Nov 22.
Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6.

Paine MJ, McLaughlin LA, Flanagan JU, Kemp CA, Sutcliffe MJ, Roberts GC, Wolf CR.

Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, 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 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), 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.

online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12446689&dopt=Abstract

Jpn J Ophthalmol. 2002 Sep-Oct;46(5):488-95.
Effects of various eye drops on corneal wound healing after superficial keratectomy in rabbits.

Tani E, Katakami C, Negi A.

Division of Organ Therapeutics, Department of Ophthalmology, Kobe University Graduate School of Medicine, Japan.

PURPOSE: The effects of various eye drops on corneal wound healing, particularly in the subepithelial haze area, were investigated histologically following superficial keratectomy in rabbits. METHODS: Mechanical superficial keratectomy was performed in rabbit eyes. Tranilast, betamethasone, hyaluronic acid, and diclofenac eye drops were administered after the procedure. Physiological saline was used as a control. Corneas were excised 1, 2, 3, and 4 weeks after keratectomy, labeled with 3H-thymidine or 3H-proline, and subjected to autoradiography. RESULTS: In the control and diclofenac groups, corneal haze occurred 3 weeks after keratectomy. Histological examination revealed an accumulation of proliferating keratocytes and active synthesis of collagen in the subepithelial area. In the tranilast and betamethasone groups, formation of corneal haze was reduced compared to the controls. The proliferation of keratocytes and the production of collagen in the corneal stroma were inhibited by these drugs. In the hyaluronic acid group also, corneal haze was decreased. In this group, although the proliferation of keratocytes was activated compared to the controls, abnormal accumulation of keratocytes in the subepithelial area was not detected. CONCLUSIONS: Tranilast and betamethasone decrease the formation of subepithelial haze by inhibiting keratocyte proliferation and synthesis of extracellular matrix in the corneal stroma. Hyaluronic acid, on the other hand, inhibits subepithelial haze by promoting physiologic wound healing.

online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12457906&dopt=Abstract

Arch Biochem Biophys. 2003 Jan 1;409(1):80-91.
Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid.

Melet A, Assrir N, Jean P, Pilar Lopez-Garcia M, Marques-Soares C, Jaouen M, Dansette PM, Sari MA, Mansuy D.

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Paris V, 45 Rue des Saints-Peres, 75270 06 Paris Cedex, France.

A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.

online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12464247&dopt=Abstract

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