- 學位論文
人類磷酸核糖焦磷酸合成酶Histidine193催化反應 之角色
The Role of Histidine193 in Human Phosphoribosyl Pyrophosphate Synthetase Catalyzed Reaction
摘要
Human phosphoribosyl pyrophosphate synthetase 1(hPRS1: EC 2.7.6.1)催化ATP和R5P (ribose 5-phosphate)反應形PRPP (phosphoribosyl pyrophosphate)與AMP。PRPP 是生物中體內重要的代謝物質, 参與嘌呤、嘧啶核甘酸合成和吡啶的生合成及回收路徑。研究指出，PRS分為三種類型，人類的PRS屬於第一類型，需要Mg2+ 與磷酸根幫助其催化，且會透過ADP進行異位調控抑制作用。目前在人類中已發現許多與PRS缺陷相關的疾病，PRS活性過高導致嘌呤核甘酸與PRPP加速生成尿酸進而形成痛風，而活性降低時則導致周邊神經病變、視神經病變、智力不足。 根據臨床上顯示hPRS1 superactivity的病患中發現PRS突變型H193L，為了解H193L突變型在人類PRS中所扮演PRS催化之機制並與人類野生型PRS的活性來比較。hPRS1結構是由三個同源二聚體組成具功能的同源六聚體，其活化位包含ATP及R5P的結合位，而磷酸根則是作為活化子與ADP競爭在異位調控位上，Asp183是位於C-domain二聚體界面與相鄰亞基的His193形成氫鍵。因此我們應用定點突變方式並利用E.coli大量表達人類wild-type及H193L突變型PRS，再利用鎳離子親和性層析管柱來純化，並應用Couple assay測得PRS反應速率，最後利用穩定態動力學來探討突變在催化反應中扮演的角色並進一步以螢光，來探討H193L突變型在二級結構和局部構型的變化。從初速度結果，獲得H193L突變型PRS降低的催化常數。實驗結果符合ordered bi bi的動力學機制，同時WT會受到MgATP受質的抑制。H193L的突變破壞二聚體界面間的氫鍵，因而增加flexible catalytic loop (K197-V202)移動到催化位置而導致活性降低。
關鍵字
磷酸核糖焦磷酸合成酶並列摘要
Human phosphoribosyl pyrophosphate synthetase 1(hPRS1: EC 2.7.6.1) catalyzes the synthesis of phosphoribosyl pyrophosphate (PRPP) from ATP and ribose 5-phophate (R5P). hPRS1 requires inorganic phosphate (Pi) and Mg2+ as activators in a reaction, but is inhibited allosterically by ADP. PRPP is a key intermediate of metabolism for biosynthesis of the purine and pyrimidine nucleotides, the pyridine nucleotide cofactors NAD+ and NADP, and the amino acids histidine and tryptophan. Mutant of H193L hPRS1 shows a superactivity in patient resulting in an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction. The crystal structure of hPRS1 is homohexamer, where Asp183 located at the dimer interface of one subunit is involved in a hydrogen bond to His193 of the adjacent subunit in the homodimer. Hence we study the role of H193 in the hPRS1 catalyzed reaction to explore its function. We have cloned, expressed, and purified H193L mutant of hPRS1. The activity was measured by a coupled assay with adenylate kinase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product AMP to the conversion of NADH to NAD+. The change in the secondary structure and local conformation is studied by protein fluorescence. The initial rates exhibited a hyperbolic dependence on the concentration of R5P in the presence of 50 mM phosphate. The kinetic parameters of kcat, kcat/Km and KR5P for H193L mutant enzyme at pH 7.5 are 2.65 s-1, 0.049 ?嵱-1s-1?nand 54 ?嵱, respectively. The kinetic parameters of H193L mutant enzyme decrease by 24-fold in kcat, 39-fold in kcat/Km, and increase 1.6-fold in KR5P compared to those parameters of the wild-type enzyme. These results indicate mutation of H193 to leucine in hPRS1 causes the decrease in catalytic constant and the decrease in affinity with R5P.