- 作者: 潘福; 李賢雄; 王仙宇
- 作者服務機構: 國防醫學院生物化學研究所; 榮民總醫院醫學研究部
- 中文摘要:
麩胺醯胺、麩胺酸及精胺酸三種胺酸活化?的動力學反應機轉,已知的全屬於連續型,必需有tRNA時,才能催化
ATP-PPi交換反應,有別於其他胺酸活化?的交替型機轉。但我們發現人體胎然麩胺醯胺活化?的性質相當特殊,並不
需要tRNA就能催化[32p]ppi-ATP交換反應。進一步綜合各項實驗結果,我們乃對該?提出〝兩步驟連續型〞動力學機轉
,以tRNA為領先基質,繼以不定序加上ATP與胺酸,先生成與?結合的中間物-胺醯腺?酸,然後依序釋出AMP, PPi
與胺醯核酸三種反應產物。l - 英文摘要: The order of interaction of substrates and products with human placental glutaminyl-tRNA synthe-tase was investigated in the aminoacylation reaction by using the steady-state kinetic methods. The initialvelocity patterns obtained from both the glutamine-ATP and glutamine-tRNA substrate pairs were inter-secting, whereas ATP and tRNA showed double competitive substrate inhibition. Dead-end inhibitionstudies with an ATP analog, tripolyphosphate, showed uncompetitive inhibition when tRNA was the vari-able substrate. The product inhibition studies revealed that PPi was an uncompetitive inhibitor with respectto tRNA. The noncompetitive inhibition by AMP versus tRNA was converted to uncompetitive by increas-ing the concentration of glutamine from 0.05 to 0.5 mM. These and other kinetic patterns obtained fromthe present study, together with our earlier finding that this human enzyme catalyzed the ATP-PPi ex-change reaction in the absence of tRNA, enable us to propose a unique two-step, partially ordered sequen-tial mechanism, with tRNA as the leading substrate, followed by random addition of ATP and glutamine.The products may be released in the following order: AMP, PPi and then glutaminyl-tRNA. The proposedmechanism involves both a quarternary complex including all three substrates and the intermediary forma-tion of an enzyme-bound aminoacyl adenylate, common to the usual sequential and ping-pong mechanisms,respectively, for other aminoacyl-tRNA synthetases.
- 中文關鍵字: amino acid; glutaminyl-tRAN; human; kinetic mechanism; synthetase
- 英文關鍵字: --