CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability

doi:10.1016/S0006-291X(02)00328-5

Biochemical and Biophysical Research Communications

Volume 293, Issue 3, 10 May 2002, Pages 969-973

https://doi.org/10.1016/S0006-291X(02)00328-5 Get rights and content

Abstract

Comparing bufuralol 1^′-hydroxylase activity among liver microsomes prepared from individuals whose CYP2D6 genotypes had been determined, we found that the activity tended to decrease depending on the number of the CYP2D6*10 allele. Pre-incubation of liver microsomes from individuals homozygous for the CYP2D6*10 allele resulted in a decrease in the enzyme activity more rapidly than those from individuals homozygous for the CYP2D6*1, suggesting that not only the catalytic activity but also the thermal stability of the enzyme appeared to be affected by the genetic polymorphism. To confirm this hypothesis, the kinetic parameters of CYP2D6.1 and CYP2D6.10 were compared for bufuralol 1^′-hydroxylation and dextromethorphan O-demethylation using microsomes prepared from yeast transformed with plasmids carrying CYP2D6 cDNAs (*1A and *10B). Kinetic studies of these CYP2D6 forms indicated clear differences in the metabolic activities between the wild (CYP2D6.1) and the mutant enzymes (CYP2D6.10). Bufuralol 1^′-hydroxylase activity in microsomes of yeast expressing CYP2D6.10 was rapidly decreased by heat treatment, supporting the idea that the thermal stability of the enzyme was reduced by amino acid replacement from Pro (CYP2D6.1) to Ser (CYP2D6.10). These data strongly suggest that the thermal instability together with the reduced intrinsic clearance of CYP2D6.10 is one of the causes responsible for the known fact that Orientals show lower metabolic activities than Caucasians for drugs metabolized mainly by CYP2D6, because of a high frequency of CYP2D6*10 in Orientals.

Section snippets

Materials and methods

Chemicals and reagents. NADP⁺, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase were obtained from Oriental Yeast (Tokyo, Japan). (±)-Bufuralol hydrochloride, (±)-hydroxybufuralol maleate, dextromethorphan hydrobromide, dextrorphan hydrochloride, 3-methoxymorphinan hydrochloride and the MAB-2D6/Human CYP2D6 WB kit were obtained from Daiichi Pure Chemicals (Ibaraki, Japan). CuOOH was obtained from Aldrich (Milwaukee, WI). All other chemicals and solvents were of the highest grade

Variation of catalytic activity of CYP2D6 in human liver microsomes

We determined the genotype of 17 human subjects whose liver samples were used for assays. As shown in Table 1, the rate of bufuralol 1^′-hydroxylase activity in liver microsomes prepared from individuals possessing the CYP2D6*10 allele tended to be lower than those in microsomes from individuals homozygous for the CYP2D6*1A. Present data indicate that a decrease in catalytic activity appears to be dependent on the number of the CYP2D6*10 allele, demonstrating the existence of a gene–dosage

Discussion

A proline-rich region is present following the signal-anchor sequence in the amino terminal portion of all known microsomal CYPs. In CYP2D6, the catalytic domain linking with the N-terminal signal-anchor region contains a proline-rich region. The sequence of PPGP is highly conserved in CYP2 family [12]. The functional role of the PPGP region in CYPs remains unclear. The substitution of only one proline residue to alanine in the proline-rich region resulted in the complete loss of heme

Acknowledgements

This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan and also by a Grant (No. 99-2) from the Organization for Pharmaceutical Safety and Research (OPSR). K. N. was supported by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. We thank Dr. N. Kagawa, Dr. I.H. Hanna and Dr. K. Kusano (Vanderbilt University) for helpful advice with this study.

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^☆: Abbreviations used: CuOOH, cumene hydroperoxide; CYP, cytochrome P450; EM, extensive metabolizer; HPLC, high-performance liquid chromatography; MAB, monoclonal antibody; MR, metabolic ratio; PM, poor metabolizer; PPGP, proline–proline–glycine–proline; PXXP, proline–an amino acid–an amino acid–proline; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; WB, Western blotting.

¹: Present address: Department of Clinical Pharmacy, Chiba University School of Medicine, Chiba 263-8522.

²: Present address: Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934.

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CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability☆

Abstract

Section snippets

Materials and methods

Variation of catalytic activity of CYP2D6 in human liver microsomes

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature

Pharmacogenetics