Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase.
Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase.
复制标题
DOI：
10.1021/acs.biochem.8b00559
复制
发表时间：
2018-07-24
期刊：
影响因子：
2.9
通讯作者：
da Silva RG
中科院分区：
文献类型：
--
作者：
Fisher G;Thomson CM;Stroek R;Czekster CM;Hirschi JS;da Silva RG
短型ATP磷酸核糖基转移酶（ATPPRT）是一种异八聚体变构酶，包括四个催化亚基（HisGS）和四个调节亚基（HisZ）。ATPPRT催化ATP和5-磷酸-α-d-核糖基-1-焦磷酸（PRPP）在Mg ~（2+）的作用下缩合生成N1-（5-磷酸-β-d-核糖基）-ATP（PRATP）和焦磷酸，这是组氨酸生物合成的第一步反应。虽然HisGS本身具有催化活性，但在不存在组氨酸的情况下，其活性被HisZ变构增强。在组氨酸存在下，HisZ介导ATPPRT的变构抑制。在这里，初始速度模式，等温滴定量热法，和差示扫描荧光法建立一个独特的动力学机制ATPPRT PRPP是第一个基板结合。AMP是HisGS的抑制剂，但稳态动力学和31 P NMR光谱表明ADP是替代底物。置换Mg 2 + Mn 2+增强催化HisGS，但不是全酶，这表明不同的限速步骤的非活化和活化的酶的形式。密度泛函理论计算证明了由两个当量的金属离子稳定的类SN 2过渡态。自然键轨道电荷分析指出Mn 2+通过在过渡态更有效的电荷稳定来增加HisGS反应速率。高溶剂粘度增加了HisGS的催化速率，但降低了杂八聚体的催化速率，表明化学和产物释放分别是HisGS和ATPPRT的速率限制。这通过前稳态动力学证实，其中用异源八聚体而不是用HisGS观察到产物形成的爆发。这些结果与激活机制一致，其中HisZ结合导致HisGS的更具活性的构象，加速化学反应超过产物释放速率。
Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.
登录
查看更多内容
影响因子：
4.1
作者：
Kulis-Horn, Robert K.;Persicke, Marcus;Kalinowski, Joern
通讯作者：
Kalinowski, Joern
影响因子：
5.7
作者：
Kulis-Horn RK;Persicke M;Kalinowski J
通讯作者：
Kalinowski J
DOI：
10.1073/pnas.1717621115
发表时间：
2018-03-20
影响因子：
11.1
作者：
Fan, Yifei;Cross, Penelope J.;Parker, Emily J.
通讯作者：
Parker, Emily J.
影响因子：
3.9
作者：
KLEEMAN, JE;PARSONS, SM
通讯作者：
PARSONS, SM
影响因子：
2.9
作者：
Champagne, Karen S.;Piscitelli, Elise;Francklyn, Christopher S.
通讯作者：
Francklyn, Christopher S.