The Reaction of Fumarase with Iodoacetate and 4-Bromocrotonate

The reaction of fumarase with several alkylating agents has been examined. The structural similarity between these compounds and the natural substrates for the enzyme, fumarate and malate, suggested that reaction might occur with structures in the active sites. Of all the compounds tested, only iodoacetate and 4-bromocrotonate inactivated fumarase by modification of structures other than the thiol groups. Because the thiol groups appear to fulfill a structural role in the enzyme, it is possible that iodoacetate and bromocrotonate react specifically with fumarase. The enzyme is inactivated to an extent of 80 to 90% when only 3 to 6 eq of each compound have reacted with the enzyme. Inactivation by each compound is reduced markedly by substrates and competitive inhibitors, and extensive structural changes do not occur after inactivation. S-Carboxymethylmethionine and 3-carboxymethylhistidine are the major derivatives found after inactivation with iodoacetate. The distribution of these carboxymethyl derivatives among the tryptic peptides of iodoacetate-inactivated fumarase has been examined. From 80 to 85% of the S-carboxymethylmethionine and 3-carboxymethylhistidine present in the modified enzyme have been recovered in three unique peptides.