MacRae I, Segel I H
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Arch Biochem Biophys. 1997 Jan 1;337(1):17-26. doi: 10.1006/abbi.1996.9751.
Fungal ATP sulfurylase has been reported to be allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the product of adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the sulfate activation sequence. However, the affinity of ATP sulfurylase for its immediate product, APS, is 1000 times higher than that for PAPS. Moreover, each sulfurylase subunit contains two sulfonucleotide binding sites (the catalytic site and a C-terminal, APS kinase-like allosteric site). Consequently, the possibility that the cooperative effects were caused solely by trace levels of APS, or by APS acting in concert with PAPS could not be dismissed. To identify the true allosteric effector, the molybdolysis reaction kinetics in the absence and in the presence of APS kinase were compared. The rationale was that in the absence of APS kinase, submicromolar levels of APS would be generated from contaminating SO(2-)4 present in the assay components, while in the presence of APS kinase, any APS formed would be converted to PAPS. The results were as follows: In the presence of added APS kinase, the initial velocity versus [MgATP] or versus [MoO(2-)4] plots at 100 microM PAPS were clearly sigmoidal as was the velocity versus [PAPS] plot at subsaturating substrate levels. Hill coefficients were in the range of 2 to 3. Also, low concentrations of S2O(2-)3offn inhibitor competitive with MoO(2-)4, activated the reaction at high PAPS and low substrate levels. These results are consistent with PAPS serving as a classical allosteric inhibitor. Although APS kinase should be superfluous to the molybdolysis reaction, the omission of this enzyme from assay mixtures resulted in rates that were higher, the same as, or lower than the corresponding "plus APS kinase" rates, (depending on the fixed level of substrates and PAPS). Additionally, the "minus APS kinase" velocity curves were less sigmoidal and, in some cases, nearly hyperbolic. The effect of APS kinase was shown to be catalytic in nature. If the data are analyzed in terms of the concerted transition (symmetry) model for allosteric enzymes, the cumulative experimental results indicate that PAPS is the true allosteric inhibitor of fungal ATP sulfurylase, binding preferentially to the T-state allosteric site (or to the allosteric site of the R state inducing the R --> T transition), while APS binds preferentially to the R state, probably as a competitive product inhibitor at the catalytic site. If it is assumed that occupancy of the allosteric site by any ligand that fits would induce the R --> T transition, then the results suggest that the allosteric site has evolved to have a higher affinity for PAPS than for APS (in contrast to real APS kinase). Computer-assisted simulations allowing for APS and PAPS binding to both the catalytic and regulatory sites of the hexameric enzyme yielded results that nearly duplicated the experimental curves.
据报道，真菌ATP硫酸化酶受到3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）的变构抑制，PAPS是腺苷5'-磷酸硫酸酯（APS）激酶的产物，而APS激酶是硫酸盐活化序列中的第二种酶。然而，ATP硫酸化酶对其直接产物APS的亲和力比对PAPS的亲和力高1000倍。此外，每个硫酸化酶亚基包含两个硫核苷酸结合位点（催化位点和一个C末端的、类似APS激酶的变构位点）。因此，不能排除协同效应仅由痕量水平的APS或由APS与PAPS协同作用引起的可能性。为了确定真正的变构效应物，比较了在不存在和存在APS激酶的情况下钼酸分解反应动力学。其基本原理是，在不存在APS激酶的情况下，检测成分中存在的污染性SO(2-)4会产生亚微摩尔水平的APS，而在存在APS激酶的情况下，形成的任何APS都会转化为PAPS。结果如下：在添加了APS激酶的情况下，在100 microM PAPS时，初始速度与[MgATP]或与[MoO(2-)4]的关系图明显呈S形，在底物水平不饱和时速度与[PAPS]的关系图也是如此。希尔系数在2到3的范围内。此外，低浓度的与MoO(2-)4竞争的S2O(2-)3offn抑制剂在高PAPS和低底物水平下激活了反应。这些结果与PAPS作为经典变构抑制剂一致。尽管APS激酶对于钼酸分解反应应该是多余的，但从检测混合物中省略该酶会导致速率更高、相同或更低（取决于底物和PAPS的固定水平）。此外，“减去APS激酶”的速度曲线的S形较小且在某些情况下几乎是双曲线的。结果表明APS激酶的作用本质上是催化性的。如果根据变构酶的协同转变（对称）模型分析数据，累积的实验结果表明PAPS是真菌ATP硫酸化酶的真正变构抑制剂，它优先结合T态变构位点（或结合到R态的变构位点诱导R→T转变），而APS优先结合R态，可能作为催化位点的竞争性产物抑制剂。如果假设任何适合的配体占据变构位点会诱导R→T转变，那么结果表明变构位点对PAPS的亲和力比对APS的亲和力更高（与真正的APS激酶相反）。允许APS和PAPS结合到六聚体酶的催化和调节位点的计算机辅助模拟产生的结果几乎与实验曲线一致。
