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The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent.
Biochem Pharmacol. 2005 Oct 15; 70(8):1253-61.BP

Abstract

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1'-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783-92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V(max) for bufuralol 1'-hydroxylation, while the K(m) was hardly affected. The V(max) for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K(m) was much larger. For dextromethorphan, in addition to dextrorphan (O-demethylation) and 3-methoxymorphinan (N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent.

Authors+Show Affiliations

LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, HV Amsterdam, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16135359

Citation

Lussenburg, Barbara M A., et al. "The Role of Phenylalanine 483 in Cytochrome P450 2D6 Is Strongly Substrate Dependent." Biochemical Pharmacology, vol. 70, no. 8, 2005, pp. 1253-61.
Lussenburg BM, Keizers PH, de Graaf C, et al. The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent. Biochem Pharmacol. 2005;70(8):1253-61.
Lussenburg, B. M., Keizers, P. H., de Graaf, C., Hidestrand, M., Ingelman-Sundberg, M., Vermeulen, N. P., & Commandeur, J. N. (2005). The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent. Biochemical Pharmacology, 70(8), 1253-61.
Lussenburg BM, et al. The Role of Phenylalanine 483 in Cytochrome P450 2D6 Is Strongly Substrate Dependent. Biochem Pharmacol. 2005 Oct 15;70(8):1253-61. PubMed PMID: 16135359.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent. AU - Lussenburg,Barbara M A, AU - Keizers,Peter H J, AU - de Graaf,Chris, AU - Hidestrand,Mats, AU - Ingelman-Sundberg,M, AU - Vermeulen,Nico P E, AU - Commandeur,Jan N M, PY - 2005/05/09/received PY - 2005/06/30/revised PY - 2005/07/01/accepted PY - 2005/9/2/pubmed PY - 2005/12/13/medline PY - 2005/9/2/entrez SP - 1253 EP - 61 JF - Biochemical pharmacology JO - Biochem Pharmacol VL - 70 IS - 8 N2 - The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1'-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783-92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V(max) for bufuralol 1'-hydroxylation, while the K(m) was hardly affected. The V(max) for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K(m) was much larger. For dextromethorphan, in addition to dextrorphan (O-demethylation) and 3-methoxymorphinan (N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent. SN - 0006-2952 UR - https://www.unboundmedicine.com/medline/citation/16135359/The_role_of_phenylalanine_483_in_cytochrome_P450_2D6_is_strongly_substrate_dependent_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-2952(05)00454-5 DB - PRIME DP - Unbound Medicine ER -