Seubert P A, Hoang L, Renosto F, Segel I H
Arch Biochem Biophys. 1983 Sep;225(2):679-91. doi: 10.1016/0003-9861(83)90079-6.
Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.
据报道，产黄青霉的同源ATP硫酸化酶对其生理性无机底物硫酸根的活性极低。这种低活性是由5'-腺苷硫酸（APS）的强效产物抑制作用导致的假象（Ki小于0.25微摩尔）。基于将35SO4(2-)中的35S掺入可被活性炭吸附的[35S]APS的测定，即使在存在大量过量无机焦磷酸酶的情况下，也随时间呈非线性。然而，在存在过量APS激酶（以及过量焦磷酸酶）的情况下，ATP硫酸化酶反应随时间呈线性，该酶的比活性（Vmax）为6至7单位毫克蛋白-1，对应于至少400分钟-1的活性位点周转数。一价含氧阴离子如NO3-、ClO3-、ClO4-和FSO3-与硫酸根（或钼酸根）竞争，并且相对于MgATP基本无竞争性。然而，硫代硫酸根（SSO3(2-)），一种真正的硫酸根类似物和该酶的终产物抑制剂（与硫酸根或钼酸根竞争），对MgATP表现出明显的非竞争性抑制。此外，在钼酸分解测定中，APS与MgATP和钼酸根均竞争。这些结果表明：（a）正常正向反应的机制可能是随机的而非有序的；（b）一价含氧阴离子对E X MgATP复合物的亲和力比对游离E的亲和力大得多。在这方面，FSO3-、ClO4-等不是真正的硫酸根类似物，尽管它们可能模拟当MgATP处于活性位点时形成的酶结合物种。基于积分速率方程的“平均速度”图分析了非线性的ATP硫酸化酶反应进程曲线（在过量焦磷酸酶存在下APS积累或在过量APS激酶存在下PPi积累）。这种新方法对于在底物浓度没有显著变化的反应过程中受到强效产物抑制的酶很有用。分析表明，ATP硫酸化酶的内在比活性为6至7单位毫克蛋白-1。因此，APS激酶对硫酸化酶活性的明显刺激是由于抑制性APS的持续去除，而不是由于两种硫酸根激活酶的结合形成“3'-磷酸-5'-腺苷硫酸合成酶”复合物，其中硫酸化酶具有增加的催化活性。进程曲线分析表明，APS与MgATP和硫酸根均竞争，而MgPPi对两种底物均为混合型抑制剂。累积数据表明正向反应的序列是随机的，APS释放是部分限速步骤。
