枯草桿菌中磷酸核糖焦磷酸合成酶的調控與 高度保留性之天門冬胺酸特性探討

Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis

許敬鑫(Ching-Hsin Hsu)

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(2011年)

摘要

磷酸核糖焦磷酸(phosphoribosylpyrophosphate，PRPP) 對於核苷酸生合成代謝扮演非常重要的角色。PRPP synthetase 催化核糖-5-磷酸(ribose 5-phosphate, R5-P) 與ATP反應形成PRPP與AMP。從五碳糖磷酸路徑分支出來的PRPP會參與嘌呤與嘧啶核苷酸生合成以及回收路徑，進而生合成組胺酸、色胺酸與嘧啶核苷酸輔酶。 MgATP複合體作為PRPP synthetase參與反應的受質。 此外，磷酸根與鎂離子作為活化子增加細菌與哺乳動物PRPP synthetase 的催化活性，而硫酸根也與磷酸根同樣具有活化子的作用，兩者皆在高濃度的條件下,會競爭鍵結在R5-P接合位,表現出抑制效果。
透過序列比對及結構分析說明磷酸核糖焦磷酸結合區域是位於胺基酸序列天門冬胺酸-223到蘇胺酸-231。為了解枯草桿菌PRPP synthetase 中高度保留性胺基酸，天門冬胺酸-133、223、224以及227的功能性，我們採用定點突變透過穩定態動力學反應分析來探討催化情形，以及利用圓偏振二旋圖譜與螢光光譜分析結構特性。D133N、D223N、D224N與D227N的PRPP synthetase突變型在E.coli中被大量表達，並使用鎳離子親和層析管柱,純化酵素。在圓偏振二旋圖譜顯示野生型均與D223N、D224N與D227N突變型酵素二級結構呈現相似。我們以螢光光譜偵測對野生型與突變型D133N、D223N、D224N、D227N酵素的脫輔基酶型。當PRPP synthetase以285 nm激發時會觀測到酵素的內生性螢光，酪胺酸螢光在野生型、突變型D223N與D227N在306 nm 有一螢光最大吸收。突變型D133N觀察到具有tyrosinate moieties螢光吸收在337 nm，以及在突變型D224N觀察到雙峰波形具有酪胺酸與tyrosinate的螢光吸收。PRPP synthetase在結構上是屬於同源六聚體，根據酵素動力學參數顯示野生型PRPP synthetase會受到活化子磷酸根與硫酸根的調控而影響受質R5-P、Mg2+及MgATP的結合與催化，在活化子硫酸根及磷酸根下，對R5-P的Km分別為50及38μM，kcat分別25與65 s-1。在沒有活化子對Mg2+的Km為1.2 mM，在活化子硫酸根及磷酸根下，對Mg2+的Km分別為0.4及0.1mM ，活化子硫酸根與磷酸根相較沒有活化子對Mg2+其kcat分別上升8與12倍。在沒有活化子對MgATP的S0.5為38μM，活化子硫酸根及磷酸根對MgATP的S0.5分別為55及47μM，活化子硫酸根與磷酸根相較沒有活化子對MgATP其kcat分別上升3與5倍。PRPP synthetase為異位調控酵素，對MgATP時為正向協同作用，活化子硫酸根及磷酸根影響其合作性，導致nH從沒有活化子條件時的6，透過活化子的調控變為2。突變型酵素對Mg2+濃度變化之動力學探討，可觀察到到其Km相對野生型酵素上升而kcat都有明顯下降的情況，說明了這些突變胺基酸參與Mg2+結合及催化的角色。我們發現重組的PRPP synthetase具有多重催化反應:分別為正常條件下的R5-P與MgATP、單獨MgATP水解速度及PRPP synthetase本身促使NADH反應。

關鍵字

磷酸核糖焦磷酸合成酶枯草桿菌 ； 調控

並列摘要

Phosphoribosylpyrophosphate (PRPP) is an important metabolite essential for nucleotide synthesis. PRPP synthetase (EC 2.7.6.1) catalyzes the reaction of ribose 5-phosphate with ATP to yield PRPP and AMP, links the pentose phosphate pathway to the pyrimidine and purine nucleotide de novo and salvage pathways, the biosynthesis of histidine and tryptophan, and pyridine nucleotide coenzymes. MgATP acts as the actual substrate of the enzyme. In addition, phosphate and Mg2+ usually act as activators for the activity of bacterial and mammalian PRPP synthetases although SO42- can mimic the effect of phosphate. Both phosphate and SO42- at high concentrations can exhibit an inhibitory effect due to their competitive binding at the R5-P binding site. Sequences alignment suggests that phosphoribosylpyrophosphate binding fingerprint motif is located at residues Asp 223- Thr 231. To investigate the functions of the conserved residues Asp-133, Asp-223, Asp-224, and Asp-227 of Bacillus subtilis PRPP synthetase, we performed site-direct mutagenesis to study the catalysis through the analysis of the steady state kinetics of the reaction and the structural analysis by circular dichroism and the fluorescence spectrometer. Mutants of D133N, D223N, D224N, and D227N were generated, overexpressed in the Escherichia coli strain BL21(DE3) and purified by Ni+ affinity column to homogeneity. The wild-type and mutants of D223N, D224N and D227N show a similar secondary structure based on circular dichroism spectra. To further probe whether the conformation of PRPP synthetase is affected by the mutation on the ribose 5-phosphate binding site, we measured the fluorescence spectrum for the apoenzymes of wild type and mutants of D133N, D223N, D224N, and D227N. The tyrosine fluorescence spectra of wild-type, D223N, and D227N exhibited a single emission λmax at 306nm when excited at 285nm. While tyrosinate moieties fluorescence emission at λmax 337nm was observed for D133N mutant and a double-humped form with both tyrosine and tyrosinate emission spectra was observed for D224N mutant. Steady-state kinetic study shows the activity of PRPP synethetase is regulated by phosphate and sulfate which affects the binding of R 5-P, Mg2+ and MgATP and catalysis. The kinetic parameters for the R5-P in the presence of sulfate and phosphate are 50 and 38 uM in Km, and 25 and 65 s-1 in kcat, respectively. The kinetic parameters Km and kcat of Mg2+ is 1.2mM and 4.9 s-1 in the absence of activator, increase to 0.4 and 0.1mM in Km, and 8 and 12 folds in kcat in the presence of sulfate and phosphate, respectively. The kinetic parameters the S0.5 and kcat of MgATP is 38μM and 10 s-1 in the absence of activator, increase to 55 and 47μM in Km, and 3 and 5 folds in kcat in the presence of sulfate and phosphate, respectively. PRPP synthetase is allosteric enzyme, shows positive cooperativity of MgATP. An addition of the activator sulfate or phosphate affects its cooperativity, resulting from 6 down to 2 nH. Mutant enzymes of D133N, D224N, and D227N increase Km-Mg2+ and decrease kcat. These results indicate that the residues D133, D224 and D227 in the active site play an important role in PRPP synthetase-catalyzed reaction. Finally, we observed recombinant PRPP synthetase has a multiple catalytic promiscuity.

並列關鍵字

PRPP synthetase ； Bacillus subtilis ； regulation

參考文獻

1. Arnvig, K., Hove-Jensen, B., and Switzer, R.L. (1990). Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis. Eur J Biochem 192, 195-200.

2. Arts, W.F., Loonen, M.C., Sengers, R.C., and Slooff, J.L. (1993). X-linked ataxia, weakness, deafness, and loss of vision in early childhood with a fatal course. Ann Neurol 33, 535-539.

3. Eads, J.C., Scapin, G., Xu, Y., Grubmeyer, C., and Sacchettini, J.C. (1994). The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. Cell 78, 325-334.

4. Eriksen, T.A., Kadziola, A., Bentsen, A.K., Harlow, K.W., and Larsen, S. (2000). Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 7, 303-308.

5. Eriksen, T.A., Kadziola, A., and Larsen, S. (2002). Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis. Protein Sci 11, 271-279.