Studies on chemical and enzymatic synthesis of maleyl-CoA.

In the course of the enzymatic reaction of acetoacetyl-CoA with maleate, catalyzed by CoA transferase, a transient appearance of free CoA-SH occurred. Subsequently, both free CoA and acyl-CoA decreased with time, indicating the formation of an unusual CoA derivative resistant to alkaline hydrolysis. During the chemical reaction of CoA-SH with maleic anhydride, the SH groups of CoA disappeared quickly, but not more than 30% could be accounted for as thioester. The product is unstable at neutrality and is hydrolyzed by nitroprusside reagent. Another product having an acyl bond of low reactivity, which reacts with hydroxylamine and does not undergo ammonolysis, but is susceptible to alkaline hydrolysis, slowly accumulated, accounting for about 25% of the CoA that disappeared. The main product appears to be formed by the addition of CoA-SH to the double bond of maleic anhydride. Column chromatography of the products of the chemical and the enzymatic reaction revealed two products, designated at X1 and X2, showing absorbance of the adenine moiety of CoA, containing 14C-labeled maleate and no free SH groups. Alkaline hydrolysis of X1 resulted in the recovery of CoA and in an increase of X2. The results are interpreted as indicating that maleyl-CoA readily hydrolyzes and reacts spontaneously with the SH group of free CoA to form an addition compound. The thioester of this product slowly hydrolyzes to give rise to the final product, which appears to be the thioether, a stable and metabolically inert compound.