枯草桿菌中磷酸核糖焦磷酸合成酶的調控機制探討

磷酸核糖焦磷酸(phosphoribosyl pyrophosphate，PRPP)作為生物體內核苷酸合成代謝重要的一個前驅物質，參與嘌呤、嘧啶和吡啶的生合成及回收路徑，並在細菌與植物中參與色胺酸和組胺酸生合成反應。PRPP synthetase (PRS) (EC 2.7.6.1)催化核糖-5-磷酸(ribose 5-phosphate，R5-P)與ATP反應形成PRPP及AMP。人類PRS缺陷疾病中，活性增加使嘌呤含量過高導致血液及組織中尿酸過高進而產生痛風，而活性降低，造成GTP下降，導致周邊神經病變。研究指出，PRSs分為三種類型，而枯草桿菌以及人類PRS皆屬於第一類型，需要Mg2+ 與磷酸根幫助其催化，且會被ADP進行異位調控所抑制。結構上枯草桿菌PRS是由三個同源二聚體組成具功能性的同源六聚體，其活化位包含ATP及R5-P的結合位，而磷酸根及硫酸根則是作為活化子與ADP競爭在異位調控位上。為了探討枯草桿菌PRS 活化子與受質之間調控的作用，首先將枯草桿菌野生型PRS在E.coli中大量表達，並透過鎳離子親和性層析管柱進行純化，最後利用穩定態動力學反應來進行探討。在活化子的部分，結合上磷酸根或硫酸根皆會影響受質的活性與結合能力，沒有活化子的環境下，R5-P的Km為2.2 μM，在活化子磷酸根及硫酸根的環境下，R5-P的Km分別為108及50 μM，而對R5-P的kcat，在沒有活化子的環境下為5.8 s-1，相較於加入活化子磷酸根及硫酸根的kcat分別上升14倍及6倍。在缺乏活化子中高濃度R5-P活性有持續增加的現象，呈現負協同作用。而在具磷酸根條件下則有抑制的情形，因此在不同磷酸根條件下改變R5-P濃度，隨著磷酸根濃度的增加其對R5-P結合能力有下降而其活性有上升的情形，實驗結果說明枯草桿菌PRS的催化反應中活化子的作用可能為V-type活化子以及K-type抑制子；另一方面，在沒有活化子的環境下，MgATP的K0.5為38 μM，在磷酸根及硫酸根的環境下分別為101及89 μM，而相較於沒有活化子的情形下加入磷酸根及硫酸根其kcat分別上升2.6及2倍。在沒有活化子的條件下MgATP為正協同作用，其nH為4.2，而在磷酸根與硫酸根的條件下其nH分別為1.8及1.9。另外，分別在0.7、10及50 mM R5-P環境下，MgATP也呈現正協同作用，其nH分別為4.2、4.1及2.7。說明當增加R5-P、磷酸根及硫酸根濃度時，會使枯草桿菌PRS穩定在R-form的構型而減少其正協同作用。由於R5-P的增加會使MgATP減少其合作性，進一步探討缺乏活化子的情形下在不同濃度的MgATP環境對R5-P的影響。結果顯示，在低濃度的MgATP，R5-P對枯草桿菌PRS結合呈正協同作用，當提高MgATP濃度達飽和時，R5-P的結合作用則轉變成負協同作用。

關鍵字

磷酸核糖焦磷酸；酵素動力學

並列摘要

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.

並列關鍵字

Phosphoribosyl Pyrophosphate Synthetase ； enzyme kinetic

參考文獻

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枯草桿菌中磷酸核糖焦磷酸合成酶的調控機制探討

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