人類磷酸核糖焦磷酸合成酶基因選殖、表達及枯草桿菌中磷酸核糖焦磷酸合成酶的催化機制探討

Cloning and Overexpression of Human Phosphoribosyl Pyrophosphate Synthetase and Catalytic Mechanism of Phosporibosyl Pyrophosphate Synthetase from Bacillus subtilis

何家瑋(Chia-Wei Ho)

指導教授 ： 黃啟清

高雄醫學大學/醫學院/生物化學研究所/碩士(2013年)

https://doi.org/10.6832/KMU.2013.00019

摘要

磷酸核糖焦磷酸合成酶(Phosporibosyl pyrophosphate synthetase，PRS)催化ATP和R5-P (ribose 5-phosphate)反應形成產物PRPP (phosphoribosylpyrophosphate)與AMP。PRPP在生物體內參與嘌呤、嘧啶等重要的生合成的反應。在人類中已發現許多與PRS缺陷相關的疾病是由於酵素活性過高或過低所造成的，說明PRPP是生物體內重要的代謝物質。因此我們探討枯草桿菌的催化機制並與人類PRS的活性做比較。經序列比對與催化位的結構分析，發現枯草桿菌的H135與Y96為高度保留序列，在催化反應中可能扮演重要的功能。因此利用定位點突變將H135、Y96突變成H135A與Y96F，以穩定態動力學來探討突變在催化反應中所扮演的角色。因人類PRS其結晶結構和胺基酸序列都與枯草桿菌非常相似，可藉由研究枯草桿菌PRS催化反應與人類PRS做比較，來了解人類PRS突變造成活性變化機制。我們將欲表達的枯草桿菌與人類的PRS基因利用E. coli進行大量表達，再利用鎳離子親和性層析管柱來純化酵素，接著用couple assay的方法測得反應速率後代入Michaelis-Menten 方程式進行動力學上的分析，結果發現H135A與野生型相比Km上升三倍而kcat下降四千多倍，表示H135確實在催化反應中扮演重要角色。Y96F其Km上升三點五倍但kcat卻是相似的，說明Y96上的氫氧基可能是扮演與受質結合的功能。由pH profile數據得知野生型酵素是利用酸鹼來進行催化。然而H135A突變型雖然影響催化，但pH profile結果與野生型相同，因此並非扮演一般酸鹼角色。先前文獻指出H135可能參與二價金屬離子的結合。在H135A改變Mg2+濃度的結果，其Km值較野生型高2倍，說明H135A確實會稍微影響與Mg2+的結合，但其活性大幅下降原因未來還需更進一步地探討。比較人類與枯草桿菌PRS動力學，在不同活化子條件下改變R5-P濃度實驗，其Km值下降低約3倍，但kcat值是相似的，說明人類PRS對於R5-P有較高的親和性。但在沒有活化子情況下， Km上升4735多倍。因此相較於枯草桿菌，人類PRS則是需要依賴activator來幫助活化。不同活化子條件下改變MgATP濃度實驗，在無活化子、硫酸根、磷酸根的情況與枯草桿菌比較其S0.5分別下降約3倍、5倍及7倍，但kcat則是差不多的。可看出人類PRS對ATP有較高的特異性。此外在沒有活化子條件下加入高濃度R5-P時，kcat與S0.5值都變成與硫酸根的結果相似，推測高濃度R5-P可能會結合在調控位上扮演activator的功能。

關鍵字

人類磷酸核糖焦磷酸合成酶

並列摘要

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.

並列關鍵字

Human Phosphoribosyl Pyrophosphate Synthetase

參考文獻

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