Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates

Background: Porphobilinogen synthase is the second enzyme involved in the b iosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form po rphobilinogen. Each substrate is recognized individually at two different a ctive site positions to be regioselectively introduced into the product. Ac cording to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechan isms for the first bond-forming step between the two substrates have been p roposed. The first involves carbon-carbon bond formation (an aldol-type rea ction) and the second carbon-nitrogen bond formation, leading to an iminium ion. Results: With the help of kinetic studies, we determined the Michaelis cons tants for each substrate recognition site. These results explain the Michae lis-Menten behavior of substrate analog inhibitors - they act as competitiv e inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. Conclusions: Analysis of the different classes of inhibition behavior allow ed us to make a correlation between the type of inhibition and a specific s ite of interaction. Analyzing the inhibition behavior of analogs of postula ted intermediates strongly suggests that carbon-nitrogen bond formation occ urs first.