GLUTATHIONE S-TRANSFERASES ACT AS ISOMERASES IN ISOMERIZATION OF 13-CIS-RETINOIC ACID TO ALL-TRANS-RETINOIC ACID IN-VITRO

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid ( 13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is no w reported. Rates of cis-trans isomerization were determined quantitat ively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a K-m of approx. 8 mu M. The maximal velocity of the reaction and the c atalytic efficiency of GSTs were determined. The initial rate of the r eaction increased linearly as a function of enzyme concentration. Cata lysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incuba tion with guanidine (7-8M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% resp ectively. The same heat treatment did not significantly inhibit isomer ization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific ac tivity of GSTs was approx. 23- and 340-fold those of GSH and apoferrit in respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that spec ific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 mu M) demonstrated that intact protein thiols are required for the isomerase activities of GSTs.