Different enzyme kinetics during the glutathione conjugation of the four stereoisomers 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 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 mu-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 2 mM GSH), the apparent Vmax values were 1975 nmol/min x mg for (-)-anti-B[c]PhDE and about 60 nmol/min x mg for both (-)-syn- and (+)-syn-B[c]PhDE, with the corresponding Km values of 1.05 and 0.20 mM. The reaction of (+)-anti-B[c]PhDE determined by applying the Hill equation had an estimated Vmax value of 930 nmol/min x mg. On varying the concentration of GSH, linear Lineweaver-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.
Funk
M
M
Institute of Toxicology, University of Mainz, Federal Republic of Germany.
Gath
I
I
Seidel
A
A
Platt
K L
KL
Oesch
F
F
Zeller
H D
HD
D000818:Animals; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D008658:Inactivation, Metabolic; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010616:Phenanthrenes / Q000494:pharmacology*; D051381:Rats; D013237:Stereoisomerism
D001551:Benz(a)Anthracenes; D010616:Phenanthrenes; C090203:1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydrobenzo(c)phenanthrene; D005982:Glutathione Transferase; D005978:Glutathione; C002615:benzo(c)phenanthrene