Inhibition Studies of Porphobilinogen Synthase from Escherichia coli Differentiating between the Two Recognition Sites

• Published in: Chembiochem : a European journal of chemical biology 2001-05, Vol.2 (5), p.343-354
• Main Authors: Stauffer, Frédéric, Zizzari, Eleonora, Engeloch-Jarret, Caroline, Faurite, Jean-Philippe, Bobálová, Janette, Neier, Reinhard
• Format: Article
• Language: English
• Subjects: Aminolevulinic Acid - metabolism; Binding Sites; biosynthesis; Enzyme Inhibitors - metabolism; Escherichia coli - enzymology; inhibitors; intermediates; Kinetics; lyases; Porphobilinogen - metabolism; porphobilinogen synthase; Porphobilinogen Synthase - antagonists & inhibitors; Porphobilinogen Synthase - metabolism; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/1439-7633(20010504)2:5<343::AID-CBIC343>3.0.CO;2-1

Porphobilinogen synthase condenses two molecules of 5‐aminolevulinate in an asymmetric way. This unusual transformation requires a selective recognition and differentiation between the substrates ending up in the A site or in the P site of porphobilinogen synthase. Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites. The P site, whose structure is known from X‐ray crystallographic studies, tolerates ester functions well. The A site interacts very strongly with nitro groups, but is not very tolerant to ester functions. This differentiation is a central factor in the asymmetric handling of the two identical substrates. Finally, it could be shown that the keto group of the substrate bound at the A site is not only essential for the recognition, but that an increase in electrophilicity of the carbon atom also increases the inhibition potency considerably. This has important consequences for the recognition process at the A site, whose exact structure is not yet known. The use of analogues of intermediates and bisubstrates in studies on the biosynthesis of porphobilinogen provides new insights into the mechanism of porphobilinogen synthase (PBGS) (see picture; X,Y=COO−, COOCH3, NO2). The inhibition studies show the distinct recognition of the two carboxylates at the A and the P site of PBGS. The enzyme differentiates between the two identical molecules of the substrate, 5‐aminolevulinic acid, already at the stage of the formation of the Michaelis–Menten complexes before incorporating them into the product in an asymmetric fashion.