大肠杆菌dimethylallyl二磷酸:tRNA dimethylallyltransferase:重组酶的绑定机制。
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摩尔JA,普特CD
大肠杆菌dimethylallyl二磷酸:tRNA dimethylallyltransferase:重组酶的绑定机制。
生物化学。1997年1月21日,36(3):604 - 14所示。
- PubMed ID
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9012675 (在PubMed]
- 文摘
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大肠杆菌dimethylallyl二磷酸:tRNA dimethylallyltransferase (DMAPP-tRNA转移酶)催化的生物合成的第一步hypermodified A37残留在图示阅读密码子开始尿苷。miaA基因编码的酶,是过度生产和纯化中明显的同质性三个步骤通过离子交换(DE52和Mono-Q)和尺寸排阻色谱法。Affinity-tagged DMAPP-tRNA包含c端三肽转移酶alpha-tubulin抗原决定基也过度生产和纯化明显的同质性在两个步骤通过离子交换和immunoaffinity色谱法。添加c端三肽alpha-tubulin抗原决定基DMAPP-tRNA转移酶不影响酶的活性。溶解不良tRNA(法)作为衬底的DMAPP-tRNA transferase-catalyzed反应分离和纯化的overexpressing克隆miaA不足的大肠杆菌菌株。单体的活性重组大肠杆菌DMAPP-tRNA转移酶。酶把dimethylallyl一半DMAPP A37,位于相邻的反密码子溶解不良tRNA(法)。所需的酶Mg2 +活动和表现出广泛的最佳pH值。tRNA米氏常数(法)和DMAPP 96 + / - 11 nM和3.2 + / - 0.5 microM,分别Vmax = 0.83 + / - 0.02 micromol最低为1 mg-1。DMAPP-tRNA转移酶结合tRNA(社会学)的离解常数5.2 + / - 1.2海里。 In contrast, DMAPP did not bind to the enzyme in the absence of tRNA. However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine. These results suggest an ordered sequential mechanism for substrate binding.