Interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analog
| Autor: | Malcolm Moos, Carina P. Tan, Gerard M. Lacourciere, Gerard H. Edwards, Richard N. Armstrong, Vikram N. Vakharia, Diane I. Morris |
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| Rok vydání: | 1993 |
| Předmět: |
Insecta
Stereochemistry Genetic Vectors Restriction Mapping Sf9 Moths Transfection Biochemistry Chromatography DEAE-Cellulose Cell Line Substrate Specificity Hydrophobic effect chemistry.chemical_compound Animals Cloning Molecular Epoxide hydrolase Epoxide Hydrolases chemistry.chemical_classification Chromatography biology Substrate (chemistry) Active site Phenanthrenes Aziridine Recombinant Proteins Molecular Weight Kinetics Durapatite Enzyme chemistry Microsomal epoxide hydrolase Microsomes Liver biology.protein Electrophoresis Polyacrylamide Gel Hydroxyapatites Baculoviridae Mathematics Phenanthrolines |
| Zdroj: | Biochemistry. 32:2610-2616 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00061a019 |
| Popis: | A recombinant baculovirus (vEHX) encoding rat hepatic microsomal epoxide hydrolase has been constructed. Infection of Spodoptera frugiperda (Sf9) cells with the recombinant virus results in the expression of the enzyme at a level estimated to be between 5% and 10% of the cellular protein. The enzyme, which can be purified in 15% yield by a simple three-step procedure involving detergent extraction, DEAE-cellulose chromatography, and removal of the detergent on hydroxylapatite, has physical and kinetic properties very close to those of the enzyme obtained from rat liver microsomes. The interaction of the enzyme with two nitrogen-containing analogues of the substrate phenanthrene 9,10-oxide (1) was investigated in order to delineate the contributions of the oxirane group and the hydrophobic surface of the substrate to substrate recognition. The enzyme exhibits altered kinetic properties toward 1,10-phenanthroline 5,6-oxide (2) in which the biphenyl group of 1 is replaced with a bipyridyl group, suggesting that hydrophobic interaction between the complementary surfaces of the substrate and active site has an influence on catalysis. The conjugate acid of the aziridine analogue of 1, phenanthrene 9,10-imine (3), in which the oxirane oxygen is replaced with NH, has a pKa of 6.1, which allows the characterization of both the neutral and protonated aziridine (3H+) as substrate analogues for the enzyme. The pH dependence of the solvolysis reveals that 3H+ rearranges to a 65/35 mixture of 9-aminophenanthrene and 9-amino-10-hydroxy-9,10-dihydrophenanthrene 10(3)-fold faster than does 3. The neutral aziridine is a competitive inhibitor (Ki = 26 microM) of the enzyme at pH 8.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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