ESCHERICHIA-COLI FUMARASE A CATALYZED TRANSFER OF O-18 FROM C-2 AND H-2 FROM C-3 OF MALATE TO ACETYLENE-DICARBOXYLATE TO FORM O-18-LABELED AND H-2-LABELED OXALACETATE

We have found that fumarase A can catalyze the transfer of O-18 from [ 2-O-18]malate and H-2 from (3R)[3-H-2]malate to the carbon skeleton of acetylenedicarboxylate to form O-18 and H-2 labeled enol oxalacetate. Our data indicates that 33% of the O-18 mobilized from [2-O-18]malate and close to 100% of the H-2 mobilized from (3R)-[3-H-2] malate would be transferred at an infinite concentration of acetylenedicarboxylate . This is the first report of oxygen transfer by an enzyme in the hydr o-lyase class, but there have been previous reports of proton transfer by enzymes of this class. The transfer of the oxygen and proton remov ed by fumarase A in the dehydration of malate requires that these atom s remain associated with the enzyme long enough for the four carbon su bstrates to interchange in the active site. With certain assumptions, the results reported in this paper allow a rough calculation of the ra te constants for the dissociation of the oxygen and proton from fumara se A. The rate constant for the oxygen is between 1 x 10(4) and 8 x 10 (5) s(-1). Since fumarase A contains a [4Fe-4S] cluster that acts as a Lewis acid in the dehydration reaction catalyzed by fumarase A, it is likely that the oxygen is bound to the enzyme as a ligand to an iron atom in the cluster. The rate constant for the dissociation of oxygen from fumarase A compares favorably with the rate constants reported fo r water interchange on iron complexes. The rate constant for the disso ciation of the proton from fumarase A is on the order of 7 x 10(4) s(- 1). This is similar to the rate constant for the dissociation of a pro ton from a carboxylic acid but higher than the rate constants for the dissociation of protons from protonated amines, protonated imidazoles, thiols, and alcohols. If the base that removes the proton from C-3 of malate in the active site of fumarase A is not a carboxylate, the dis sociation of the proton from this group must be accelerated in some wa y by the enzyme.