PBG-synthase catalyses the asymmetrical condensation of two 5-aminolevulinic acids to produce PBG.
We are working on the PBG-synthase from E. coli CR 261. This is an enzyme with a molecular weight of 280 kD constituted of eight identical subunits. The eight subunits are postulated to be organized as four functional units each composed of a dimer of the protein. The PBGS from E. coli is Zn2+ and Mg2+ dependant and shows an optimal pH value around 8.
The active site recognizes the two substrate molecules but shows a greater affinity for the first one which binds to the enzyme covalently. The amino function of a lysine side chain reacts with the keto function of the 5-aminolevulinic acid (ALA) to form an E-iminium ion . Jordan  located this first substrate in the P-side whereas Shemin  postulated that the first substrate is the A-side ALA.
The second substrate seems not be bound covalently to the enzyme. However the keto function is also essential for a good recognition of the second substrate. The carboxylate function is important for the recognition of the A-side ALA whereas the amino group has been shown to be less important.
A complex can be postulated between the second substrate and the ZnA2+  which is essential to the enzyme activity.
Our kinetic model is based on Michaelis-Menten kinetics with a rapid formation of the Schiff base between the enzyme and the first substrate at the P-site :
The next step is the complexation of the second substrate at the A-site and then followed by the formation of the product which is the rate determining step :
We differentiate four types of inhibition :
Uncompetitive or mixed :
The inhibitors interact with the P-site (uncompetitive) or with the P-site and the A-site (mixed) of the active site of the enzyme at the same time. Mixed or uncompetitive inhibitions can not be clearly differentiated by the interpretation of kinetic data only.
The slow binding inhibitors show a time dependent reversible inhibition. The inhibitors form a sufficiently well stabilized interaction with the enzyme (e.g. stabilized Schiff base at the P-site of the enzyme) to dissociate only slowly under dialysis conditions.
Inhibitors belonging to this class interact with the A-site of the enzyme.
These inhibitors bind to the enzyme covalently at the active site either at the P-site or at the A-site of the enzyme.
The procedure for testing the inhibitors is schematically shown bellow :
4,6-dioxoheptanoic acid (1) is known to be responsible for the inhibition of PBG-synthase in the disease called tyrosinemia. Tyrosinemia has its name due to accumulation of 4,6-dioxoheptanoic acid (1) as a product of the abnormal degradation of tyrosine . Testing of 1 as inhibitor with the enzyme from E. coli. Gave the smallest inhibition constant determined so far. The Ki-value of 1.4 µM is almost a factor of 10 smaller than inhibition constants determined for the next best inhibitor. The inhibition kinetics was typical for an uncompetitive or mixed inhibitor. We interpret this behavior as a consequence of an interaction of the inhibitor 1 at the P-site and may be also at the same time at the A-site of the enzyme.
The goal of our investigation is to assess the impact of structural modifications of the 4,6-dioxoheptanoic acid (1) on the efficiency as an inhibitor of PBGS. We hope to contribute to the understanding of the role of the 1,3-diketo system in the interaction with the A-site and with the P-site of the enzyme.
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