Allosteric PRPP synthetase

Primary gout can be caused by overproduction of purine catabolites due to X-linked mutations of PRPP synthetases that render the enzyme insensitive to allosteric inhibitors. 

PRPP synthetase requires inorganic phosphate as an allosteric activator. Its activity depends on intracellular concentrations of several end products of pathways in which PRPP is substrate. These end products are purine and pyrimidine nucleotides (Figure 27-12). 

A primary site of regulation is the synthesis of PRPP. PRPP synthetase is negatively affected by GDP and, at a distinct allosteric site, by ADR Thus, the simultaneous binding of an oxypurine (eg., GDP) and an aminopurine (eg., ADP) can occur with the result being a synergistic inhibition of the enzyme. This enzyme is not the committed step of purine biosynthesis PRPP is also used in pyrimidine synthesis and both the purine and pyrimidine salvage pathways. 

Fig. 41.9. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric sites, one for ADP, the other for GDP. Glutamine phosphoribosyl amidotransferase contains adenine nucleotide and guanine nucleotide binding sites the monophosphates are the most important, although the di- and tri-phosphates will also bind to and inhibit the enzyme. Adenylosuccinate synthetase is inhibited by AMP IMP dehydrogenase is inhibited by GMP.

Previous attempts to address this problem have shown no increase in specific activity of PRPP synthetase or amidophosphoribosyitransferase under conditions of stimulation of purine production (6). But in these studies enzyme assays were conducted under activating conditions in the assay cuvette. The present study (10) demonstrates that marked changes of enzyme activity can be brought about in vivo by allosteric effects that would not be detected by routine assays and that require conditions which allow recognition of the physical and catalytic state of the enzyme in the tissue at the moment of analysis. 

It thus appears that the mutant PRPP synthetase enzyme is structurally altered in such a way that only its regulatory properties but not its catalytic properties are affected. This selective alteration proves that these two properties are located at different sites, the enzyme being allosteric. Examples are known of mutations in bacteria (18,19) and Ehrlich ascites cells (20) which altered the susceptibility of regulatory enzymes to effector mulecules. An indication for such a mutation in man has been obtained by Henderson et al in studies on fibroblasts from two patients with purine overproduction and gout, showing reduced effectiv-ness of product inhibition of purine biosynthesis (2l). 

The primary site of regulation is Carbamoyl Phosphate Synthetase II (glutamine) which is allosterically inhibited by UTP. Elevated PRPP increases the CPS-II activity to help control PRPP levels. Feedback inhibition (control) is provided by TDP inhibition of PRPP synthesis and UMP inhibition of OMP Decarboxylase. 

The regulated step of pyrimidine synthesis in humans is carbamoyl phosphate synthetase 11. The enzyme is inhibited by UTP and activated by PRPP (see Fig. 41.14). Thus, as pyrimidines decrease in concentration (as indicated by UTP levels), CPS-11 is activated and pyrimidines are synthesized. The activity is also regulated by the cell cycle. As cells approach S-phase, CPS-11 becomes more sensitive to PRPP activation and less sensitive to UTP inhibition. At the end of S-phase, the inhibition by UTP is more pronounced, and the activation by PRPP is reduced. These changes in the allosteric properties of CPS-11 are related to its phosphorylation state. Phosphorylation of the enzyme at a specific site by a MAP kinase leads to a more easily activated enzyme. Phosphorylation at a second site by the cAMP-dependent protein kinase leads to a more easily inhibited enzyme. 

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