Different enzyme kinetics during the glutathione conjugation of the four stereoisomers of the fjord-region diolepoxides of benzo[c]phenanthrene by the μ-class rat liver glutathione S-transferase HTP II

Abstract

The enzyme-catalysed conjugation of each of the four stereoisomers of trans-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[ c]phenanthrene (B[c]PhDE) with glutathione (GSH) by HTP II, a novel isolated μ-class GSH transferase from the liver of untreated rat, was studied. All four stereoisomers were substrates for GSH transferase HTP II. The enzymatic reaction shows three different types of enzyme kinetics: substrate inhibition for (−)- anti-B[c]PhDE with ( R,S,S,R)-absolute configuration, allosteric behavior using (+)- anti-B[c]PhDE with ( S,R,R,S)-absolute configuration and Henri-Michaelis-Menten kinetics with both the (−)- syn- and (+)- syn-enantiomers, with ( S,R,S,R)- and ( R,S,R,S)-absolute configuration, respectively. When the concentration of these diolepoxides was varied (using 2mM GSH), the apparent V max values were 1975 nmol/min × mg for (−)- anti-B[c]PhDE and about 60 nmol/min × mg for both (−)- syn- and (+)- syn-B[c]PhDE, with the corresponding K m , values of 1.05 and 0.20 mM. The reaction of (+)- anti-B[c]PhDE determined by applying the Hill equation had an estimated V max value of 930 nmol/min × mg. On varying the concentration of GSH, linear Line-weaver-Burk plots were obtained. No competitive effect could be observed using a mixture of (−)- anti- and (+)- anti-enantiomers, indicating that their binding sites are different and independent. It was also shown, that the binding sites of (+)- anti- and both syn-enantiomers were different and independent of each other, while there was a small effect on the binding of the syn-enantiomers caused by (−)- anti-B[c]PhDE. All products of the reaction between GSH and the dihydrodiol epoxides of benzo[ c]-phenanthrene could be resolved by HPLC and were identified and quantitated using the corresponding synthetic GSH conjugates.