DIFFERENT ENZYME-KINETICS DURING THE GLUTATHIONE CONJUGATION OF THE 4STEREOISOMERS OF THE FJORD-REGION DIOLEPOXIDES OF BENZO[C]PHENANTHRENE BY THE MU-CLASS RAT-LIVER GLUTATHIONE-S-TRANSFERASE HTP-II

The enzyme-catalysed conjugation of each of the four stereoisomers of y-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE) with glu tathione (GSH) by HTP II, a novel isolated mu-class GSH transferase fr om the liver of untreated rat, was studied. All four stereoisomers wer e 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 behav iour using (+)-anti-B[c]PhDE with (S,R,R,S)absolute configuration and Henri-Michaelis-Menten kinetics with both the (-)-syn- and (+)-syn-ena ntionmers, with (S,R,R,S)- and (R,S,R,S)-absolute configuration, respe ctively. When the concentration of these diolepoxides was varied (usin g 2 mM GSH), the apparent V-max values were 1975 nmol/min x mg for (-) -anti-B[c]PhDE and about 60 nmol/min x mg for both (-)-syn- and (+)-sy n- 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 x mg. On v arying the concentration of GSH, linear Line-weaver-Burk plots were ob tained. No competitive effect could be observed using a mixture of (-) -anti- and (+)-anti-enantiomers, indicating that their binding sites a re different and independent. It was also shown, that the binding site s of (+)-anti- and both syn-enantiomers were different and independent of each other, while there was a small effect on the binding of the s yn-enantiomers caused by (-)-anti- B[c]PhDE. All products of the react ion between GSH and the dihydrodiol epoxides of benzo[c]-phenanthrene could be resolved by HPLC and were identified and quantitated using th e corresponding synthetic GSH conjugates.