Porphyrin biosynthesis in Euglena gracilis—II. Pyrrylmethane intermediates in the enzymic cyclotetramerization of porphobilinogen

Abstract

1. 1. Under normal incubation conditions pellet and deaminase Euglena gracilis preparations, are able to form two different polypyrrole intermediates called P and D, respectively, which did not behave as substrates but as intermediates in the reaction, and significantly inhibited total porphyrins formation. It was postulated that these supematants should contain a high percentage of larger polypyrroles that compete very unefficiently for its binding site with the intermediates bound to the enzyme. This hypothesis was supported by the data obtained when different amounts of free P and D polypyrroles were added to the enzymes; as expected, increasing amounts of free intermediates increasingly inhibited total porphyrinogens formation and had also an effect on isomer composition. With the pellet enzymes, the percentage of type I increased and correspondly decreased the amount of type III, while the reverse effect was obtained with deaminase. 2. 2. Experimental evidence indicate that the enzymic polymerization of PBG takes place in a non-stop sequence on the enzyme surface; so, the possible intermediates should remain bound to the enzyme until the growing polypyrrole chain reaches its critical length and it is ready for cyclization. The interaction, with the enzymes, of the natural polypyrroles P and D formed by pellet and deaminase Euglena gracilis without liberating them from their enzymes was then studied. As expected, incubation of enzyme-bound P intermediate with pellet or deaminase and PBG, did not inhibit total porphyrins formation but enhanced it. Incubation of D intermediate with deaminase and PBG also enhanced urogen I formation although some inhibition was obtained at the highest concentrations used. D intermediate significantly inhibited porphyrins synthesis by pellet. 3. 3. Therefore, results obtained studying the interaction of free and enzyme-bound intermediates with the enzymes involved in PBG polymerization suggest: (a) that the structure of the polypyrrole formed by the pellet is that corresponding to an isomerized tetrapyrrylmethane (PAAPAPAP), and that of the polypyrrole formed by deaminase corresponds to a nonisomerized tetrapyrrylmethane (PAPAPAPA); (b) that the intermediates must be bound to the enzyme because when they are added to the enzymic systems in its natural enzyme-polypyrrole association, they behaved as normal intermediates, enhancing total porphyrinogen formation.