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

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Abstract
Succinyl-CoA:3-ketoacid coenzyme A transferase [pig heart] undergoes a biphasic reaction with 2-nitro-5-(thiocyanato)benzoate, giving .apprx. 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 2 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. Apparently the essential thiol group of the enzyme is not involved directly in catalysis and it reacts with 2-nitro-5-(thiocyanato)-benzoate by 2 pathways, to form active and inactive enzymes. This can be explained by the attack of the thiol on C to form active enzyme-SCN and the attack on S to form enzyme-SSAr, which is blocked at the active site and rapidly undergoes irreversible inactivation.