Modification and inactivation of CoA transferase by 2-nitro-5-(thiocyanato)benzoate

Succinyl-CoA:3-ketoacid coenzyme A transferase undergoes a biphasic reaction with 2-nitro-5-(thiocyanato)-benzoate, giving approximately 70% loss of activity in the initial phase. Active-site titration shows that this inactivation represents the complete loss of activity of 75 +/- 5% of the enzyme molecules. The remaining 25 +/- 5% of the active sites is protected against inactivation by methyl methanethiosulfonate and 5,5'-dithiobis(2-nitrobenzoate); this protection is removed upon treatment of the modified enzyme with dithiothreitol Values of kcat/Km for the two half-reactions catalyzed by the enzyme are the same for the native and modified enzymes on the basis of number of remaining active sites. The modified enzyme shows a smaller decrease in activity with increasing pH in the range pH 7.5-8.7 than the native enzyme. It is concluded that the "essential" thiol group of the enzyme is not involved directly in catalysis and that it reacts with 2-nitro-5-(thiocyanato)-benzoate by two pathways, to form active and inactive enzymes. This can be explained by the attack of the thiol on carbon to form active enzyme-SCN and the attack on sulfur to form enzyme-SSAr, which is blocked at the active site and rapidly undergoes irreversible inactivation.