δ-Aminolevulinic Acid Dehydratase of Rhodopseudomonas spheroides

Abstract

δ-Aminolevulinic acid dehydratase catalyzes the condensation of 2 molecules of δ-aminolevulinic acid to a pyrrole, porphobilinogen. This synthesis requires an aldol condensation, the elimination of the elements of water, and the formation of a Schiff base. Our data are consistent with the following mechanism as catalyzed by the enzyme of Rhodopseudomonas spheroides. One molecule of δ-aminolevulinic acid forms a Schiff base with the enzyme. This is followed by a nucleophilic attack, by the enzyme-δ-aminolevulinic acid anion intermediate, onto the carbonyl carbon atom of a 2nd δ-aminolevulinic acid molecule. The resulting aldol loses the elements of water, and the free amino group of the 2nd molecule of the substrate displaces the amino group of the enzyme by a transimination or transaldimination, thus forming porphobilinogen. Although both levulinic acid and ethyl levulinate form Schiff bases with the enzyme, only the free acid acts as a competitive inhibitor of δ-aminolevulinic acid. The affinity of the substrate for the enzyme is dependent on the ease of forming a Schiff base, and is also dependent on the formation of an ionic bond between the carboxyl group of the substrate and a positive charge on the enzyme at a particular distance away from the Schiff base linkage. Furthermore, the formation of heterologous pyrroles was demonstrated between levulinic acid or from its ester with δ-aminolevulinic acid. Since these pyrroles can only be formed if the levulinic acid or its ester is in Schiff base linkage with the enzyme, one can predict that the molecule of δ-aminolevulinic acid which gives rise to the acetic acid side chain in porphobilinogen is the molecule which was in Schiff base linkage with the enzyme, whereas the other molecule was either at another site or came from solution.