| Summary: | 碩士 === 高雄醫學大學 === 生物化學研究所 === 101 === Phosphoribosyl pyrophosphate (PRPP) plays the role of an important precursor on nucleotide synthesis/metabolism, which involved in de novo and salvage pathway of purine, pyrimidine and pyridine. PRPP also involves in biosynthesis of histidine and tryptophan both in plant and microbacteria. PRPP synthetase (PRS) (EC 2.7.6.1) catalyzes the reaction of ribose 5-phosphate (R5-P) and ATP to yield PRPP and AMP. Mutations caused the superactivity in human PRS type 1 (hPRS1) leads to gout with uric acid overproduction, while decrease in activity causes peripheral neuropathy results from reduced levels of GTP. In previous study, PRSs have been classified into three classes, both Bacillus subtilis and Homo sapiens PRSs are belong to class I, which require Mg2+ and phosphate for enzymatic activity, but is inhibited allosterically by ADP and possibly other nucleotides. The quaternary structure of B. subtilis PRS shows functional homohexamer consists of three homodimers. The catalytic active site, consisting of the ATP and R5-P binding sites, is located at the interface of two domains of one subunits. Phosphate and ADP bind to the same allosteric site which located at interface between three subunits of the hexamer.
To investigate the regulation of B. subtilis PRS by the activator and the substrate, we expressed B. subtilis PRS in E. coli and purified B. subtilis PRS by Ni2+ sepharose affinity chromatography column. B. subtilis PRS activity was studied by the steady state kinetics assay. PRS is an allosteric enzyme. The catalytic activity of PRS and binding affinity with substrates are affected by the activators of phosphate or sulfate. In the absence of activator, the Km for R5-P is 2.2 μM, increases to 108 and 50 μM in the presence of phosphate and sulfate, respectively, while the kcat increases 14- and 6-folds, respectively. When varying the concentration of R5-P, the initial rates exhibited a substrate inhibition at saturated phosphate and no substrate inhibition at saturated sulfate. While a gradual increase rate after an initial hyberbolic curve in the absence of activator, indicating a negative cooperativity. Both Km for R5-P and enzyme activity has increased in the presence of phosphate. The results suggest phosphate and sulfate act as V-type activator and K-type inhibitor for B. subtilis PRS catalyzed reaction. In the absence of activator, the K0.5 of MgATP is 38 μM, and the K0.5 is 101 and 90 μM in the presence of phosphate and sulfate, respectively. The kcat increase 2.6- and 2-folds in the presence of phosphate and sulfate, respectively, by varying the concentration of MgATP. The initial rates exhibited a sigmoid dependence on the concentration of MgATP, giving nH of 4.2. It changes to nH of 1.8 and 1.9 in the presence of phosphate and sulfate, respectively. The results suggest a positive cooperativity for MgATP. The initial rates also exhibited a sigmoid dependence on the concentration of MgATP at 0.7, 10, or 50 mM R5-P, giving nH of 4.2, 4.1, and 2.7, respectively, indicating a positive cooperativity. These results suggest that phosphate or sulfate behaviors as an allosteric effector stabilizing the R-form and a conformational hetrogenity of B. subtilis PRS, while R5-P at high concentration seems to have a similar function as activator that decreasing the cooperactivity by stabilizing the R-form of B. subtilis PRS.
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