| Summary: | 碩士 === 高雄醫學大學 === 生物化學研究所 === 101 === Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the reaction of ribose-5-phosphate (R5P) with ATP to yield PRPP and AMP. PRPP is a precursor for the biosynthesis of purine and pyrimidine. In human, many diseases associated with PRS defects were found. PRPP is an important metabolite of most organisms. In this study, we discuss the catalytic mechanism of PRS from Bacillus subtilis and compared the activity with human PRS. According to the crystal structure of PRS and sequence alignment, H135 and Y96 found in Bacillus subtilis are highly conserved residues and may play an important role in the catalytic reaction. We used the site-directed mutagenesis to study the catalytic mechanism and adopt coupled assay and pH profile to characterize their roles in catalytic mechanism. The kcat value of Y96F and H135A mutant enzymes decreases 1.2 and 4300-folds and the Km value increase 2.8 and 3.2-folds, respectively. The results suggest that Y96F and H135A mutants may involve in substrate binding, and H135A mutant is important in enzyme catalysis. The pH profile data shows that the wild-type enzyme is involved in the acid-base catalysis. Although, the H135A mutant is important in enzyme catalysis, it’s pH profile is similar with wild-type enzyme. Therefore, H135 residues is not involved in general acid-base catalysis. Switzer et al. has proposed that H135 involve in divalent cation binding in Salmonella typhimurium PRS. We studied the H135A mutant activity by varying the consistency Mg2+. The Km for Mg2+ increases 2-fold compared with wild-type enzyme. This result shows H135A mutant does slightly affect the binding with Mg2+. However, the significant decrease in activity for H135A mutant is needed to be further investigated. In human PRS, the kinetic parameters of Km and kcat for the R5-P are decreased by 3-fold and 1-fold, respectively, compared with bsPRS, suggesting that the human PRS has a high affinity with R5-P. However, the Km increase to 4735-fold in the absence of activator. The results suggest that the human PRS need activator to activate the enzyme activity. The kinetic parameters of S0.5 compared with bsPRS decrease 3-, 5- and 7-fold in the absence of activator and in the presence of sulfate and phosphate, respectively. The result shows that human PRS has a high affinity with MgATP. While the Km and kcat at high concentration of R5-P in the absence of activator are similar in comparsion with the result in the presence of sulfate. The results suggest that high concentrations of R5-P may bind at the allosteric site to act an activator to active the human PRS catalyzed reaction.
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