The mechanism of action and the active site of porphobilinogen deaminase has been studied extensively. The mechanism of action of porphobilinogen deaminase starts with deamination of porphobilinogen to methylene pyrrolinene, which is subject to nucleophilic attack by the carbon at the free alpha position of the terminal pyrrole(either ring two of the DPM cofactor or any of the other terminal pyrroles during chain elongation). Steric hindrance prevents chain elongation from proceeding beyond a "hexapyrrole" so the product, preuroporphyrinogen, is subsequently released by hydrolysis while the dipyrromethane cofactor stays intact(see Scheme 1). The cofactor not only acts as a reaction primer but it also limits the product to the length desired(4,5,8,9).

Site-directed mutagenesis studies have established a number of invariant residues required for full activity of the enzyme including aspartate-84 and arginines 11, 131, 132, 149, 155, 176, and 232(2,3,4,8). Cysteine-242, since the dipyrromethane cofactor is covalently attached to it, is also essential for enzyme activity(7). A deep cleft, spanning about half the width and depth of the molecule, harbors the prosthetic group at it's mouth, with considerable open space behind it, to accommodate the growing pyrrole chain. The cleft is enclosed mainly by the C-termini of the beta strands, the amino termini of the alpha helices and connecting loops. It has been established that there are three pyrrole binding in the active site of the enzyme. The C site, responsible for binding the two pyrrole rings of the cofactor, contains residues that form salt bridges with it's negative acetate and propionate side chains. Arginine-131 and -132 are especially important for initially binding the cofactor because mutagenesis of either of these residues results in an apoenzyme unable to assemble the prosthetic group. The S site, responsible for binding the substrate, porphobilinogen, is made up of arginines 11, 149, 155 and serine-13. After porphobilinogen is bound and carbon-carbon bond formation is complete, a translocation occurs allowing another molecule of porphobilinogen to bind to the S site while the terminal pyrrole moves to the C site. This translocation is dependant on polar interactions of arginine-176 and 232, positioned deeper in the catalytic cleft. Mutagenesis of these residues stops chain elongation before the third or fourth pyrrole unit can be added(1,2,4,5). Aspartate-84 has also been shown to be required for activity of the enzyme. It's carboxylic side chain is in position to hydrogen bond with the pyrrole nitrogens in the S site and the C site. The negative charge also helps to stabilize the positive charges developed during carbon-carbon bond formation of the incoming pyrrole units(2,4,8).