Rhodobacter sphaeroides phosphoribulokinase: Binary and ternary complexes with nucleotide substrate analogs and effectors
Rhodobacter sphaeroides phosphoribulokinase: Binary and ternary complexes with nucleotide substrate analogs and effectors
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DOI：
10.1021/bi9619334
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发表时间：
1996-11-26
期刊：
影响因子：
2.9
通讯作者：
Miziorko, HM
中科院分区：
文献类型：
--
作者：
Runquist, JA;Narasimhan, C;Miziorko, HM
球形红杆菌磷酸核苷酸激酶(PrK)与三磷酸腺苷底物以及光谱活性的三硝基苯基三磷酸腺苷类似物(三硝基苯基三磷酸腺苷和三磷酸腺苷-S-乙酰氨基丙基)结合，形成稳定的二元络合物。这些核苷酸三磷酸在PrK底物位置的化学计量结合不仅与野生型酶结合，而且与D42A和D169A突变体结合。这些突变体包含完整的功能底物结合位点的证明表明了它们实质上的结构完整性，并强调了它们显著降低的催化活性的重要性[Charlier等人，(1994)生物化学33,9343-9350]。类似地，PrK与变构激活剂NADH形成稳定的二元络合物。负变构效应剂AMP取代激活剂NADH，但不取代它们各自与酶的二元络合物的底物。当作为PrK底物的荧光核苷酸三磷酸腺苷与酶形成二元络合物时，其荧光发射增强，最大吸收波长从类似的557 nm移动到545 nm。当形成二元PrK-NADH络合物时，二核苷酸效应器的荧光发射也被增强，并且波长(Max)从相似的460 nm移动到440 nm。PrK形成稳定的含有NADH和ATP或三硝基苯基-ATP的三元络合物。由于能量转移，三硝基苯基-三磷酸腺苷三元络合物中的NADH荧光被显著猝灭，从而可以估计这种新的给体/受体对之间的空间分离。
Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al, (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda(max) shifted from similar to 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda(max) shifted from similar to 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.