Lansdon Eric B, Fisher Andrew J, Segel Irwin H
Department of Chemistry and Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, California 95616, USA.
Biochemistry. 2004 Apr 13;43(14):4356-65. doi: 10.1021/bi049827m.
Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration (APS) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at APS) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.
从大肠杆菌表达系统中纯化出接近均一的重组人3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）合成酶同工型1（脑型），并对其进行了动力学表征。天然酶是一种二聚体，每个71 kDa的亚基包含一个三磷酸腺苷（ATP）硫酸化酶和一个5'-磷酸腺苷硫酸酯（APS）激酶结构域，催化由ATP和无机硫酸盐整体形成PAPS。该蛋白质分离出来时就具有活性，但用二硫苏糖醇处理可增强其活性。APS激酶活性表现出典型的被APS底物抑制作用（在饱和MgATP条件下，K(I)为47.9 microM）。在APS浓度（APS）为15 microM时，观察到最大可达到的活性为0.12微摩尔·分钟⁻¹·（毫克蛋白质）⁻¹。在饱和MgATP条件下，APS的理论K(m)以及在APS条件下MgATP的K(m)分别为4.2 microM和0.14 mM。在细胞中MgATP（2.5 mM）和硫酸盐（0.4 mM）可能的水平下，在最佳测定条件下PAPS形成的整体内源性速率为0.09微摩尔·分钟⁻¹·（毫克蛋白质）⁻¹。加入过量的纯产黄青霉APS激酶后，整体速率增加到0.47微摩尔·分钟⁻¹·（毫克蛋白质）⁻¹。ATP硫酸化酶结构域的动力学常数如下：V(max,f) = 0.77微摩尔·分钟⁻¹·（毫克蛋白质）⁻¹，K(mA(MgATP)) = 0.15 mM，K(ia(MgATP)) = 1 mM，K(mB(硫酸盐)) = 0.16 mM，V(max,r) = 18.7微摩尔·分钟⁻¹·（毫克蛋白质）⁻¹，K(mQ(APS)) = 4.8 microM，K(iq(APS)) = 18 nM，以及K(mP(焦磷酸)) = 34.6 microM。（a）ATP硫酸化酶和APS激酶活性之间的失衡，（b）在整个反应过程中溶液中APS的积累，（c）外源APS激酶提供的速率加速，以及（d）两个活性位点对外源APS的可及性，所有这些都反对APS通道化。钼酸盐、硒酸盐、铬酸盐（“六价铬”）、砷酸盐、钨酸盐、氯酸盐和高氯酸盐与ATP硫酸化酶结构域结合，前五种作为替代底物促进ATP分解为AMP和PP(i)。硒酸盐、铬酸盐和砷酸盐产生短暂的APX中间体，其寿命足够长，能够被捕获并被APS激酶3'-磷酸化。（假定的PAPX产物分解为腺苷3',5'-二磷酸和原始的含氧阴离子。）氯酸盐和高氯酸盐形成无活性的E.MgATP.含氧阴离子复合物。据报道苯丙氨酸是脑ATP硫酸化酶的抑制剂，但对PAPS合成酶同工型1没有影响。
