Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro

The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoi c acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigate d the catalysis of isomerization of 13-cRA to t-RA by recombinant human glu tathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M s odium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditio ns used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1- 1 showed substrate saturation and the K-m and V-max values for the reaction were approx. 7 mu M and 650 pmol/min per nmol respectively. The reaction r ate increased linearly with increasing enzyme concentration. The reaction w as inhibited both by heat treatment and by S-decylglutathione (a potent inh ibitor of transferase activity associated with GST). Additions of polyclona l rabbit antiserum for human GSTP1-1 to the reaction resulted in a signific ant decrease in generation of t-RA (70-80 %). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomeriza tion of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction condit ions, GSTP1-1 was much less effective in catalysing the steric conversion o f 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecifi c for the conversion of 13-cRA to t-RA. These observations suggest that enz ymic catalysis was the primary mechanism for the GSTP1-1-dependent conversi on of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixt ures.