Martin R L, Daley L A, Lovric Z, Wailes L M, Renosto F, Segel I H
Department of Biochemistry and Biophysics, University of California, Davis 95616.
J Biol Chem. 1989 Jul 15;264(20):11768-75.
ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)
产黄青霉的ATP硫酸化酶是一种同型六聚体，每个亚基含有三个游离巯基，在非变性条件下，其中只有一个（称为SH-1）可被二硫键、马来酰亚胺和卤化物试剂修饰。SH-1的修饰对催化常数（kcat）影响较小，但会使MgATP和SO4(2-)（或MoO4(2-)）的半饱和底物浓度（[S]0.5）值增加一个数量级。此外，速度曲线变为S形，希尔系数（nH）约为2（雷诺斯托，F.，马丁，R.L.，和西格尔，I.H.（1987年）《生物化学杂志》262，16279 - 16288）。直接平衡结合测量证实，如果也存在硫酸盐亚位点配体（如FSO3-），[32P]MgATP以正协同方式（nH = 2.0）与SH修饰的酶结合。在没有焦磷酸亚位点配体的情况下，[35S]腺苷5'-磷酸硫酸酯（APS）与SH修饰的酶结合显示出正协同性（nH = 1.9）。结果表明，正协同性需要腺苷酰和硫酸盐（但不是焦磷酸）亚位点被占据。[35S]APS与天然酶结合显示出负协同性（或与至少两类位点结合）。[35S]APS单周转的同位素捕获曲线：（a）证实了平衡结合曲线，（b）表明六聚体的所有六个位点都具有催化活性，（c）表明APS不会以显著速率从E.APS.PPi解离。[35S]APS捕获的MgPPi浓度依赖性表明MgPPi与天然酶和SH修饰酶上的两类位点结合。在饱和APS存在下，苯乙二醛对天然酶或SH修饰酶的失活是双相的。半对数图表明只有一半的位点受到高度保护。累积数据表明存在一种模型，其中位点或亚基对可以存在于三种不同状态，分别称为HH（两个位点都具有高APS亲和力，如天然游离酶）、LL（两个位点都具有低APS亲和力，如SH修饰酶）和LH（如APS占据的天然或SH修饰酶）。因此，HH向LH的转变对APS结合显示出负协同性，而LL向LH的转变显示出正协同性。同类SH反应性试剂的相对反应活性顺序为：N-苯基马来酰亚胺大于N-乙基马来酰亚胺；二硫代硝基吡啶大于二硫代硝基苯甲酸；硫醇盐-MQ大于硫醇盐-MB。log kmod对pH的曲线表明SH-1的pKa大于9。（摘要截断于400字）
