Su Tian-Mu, Yang Yuh-Shyong
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
Biochemistry. 2003 Jun 10;42(22):6863-70. doi: 10.1021/bi0342463.
Sulfotransferase catalyzes sulfuryl group transfer between a nucleotide and a variety of nucleophiles that may be sugar, protein, xenobiotics, and other small molecules. Nucleotides may serve as cosubstrate, cofactor, inhibitor, or regulator in an enzyme catalyzed sulfuryl group transfer reaction. We are trying to understand how nucleotide regulates the activity of phenol sulfotransferase (PST) through the expression of two enzyme forms. The homogeneous rat recombinant PST was obtained from Escherichia coli, and the nucleotide copurified was examined. The nucleotide was completely removed from inactive PST in high salt and oxidative condition. Total enzyme activity was recovered following incubation in reductive environment. Many nucleotides are known to tightly bind to PST but only one nucleotide, 3'-phosphoadenosine 5'-phosphate (PAP), was identified to combine with PST by ion-pair RP-HPLC, UV-visible spectra, (31)P NMR, and ESI-MS and MS-MS spectrometry. In addition to the presence or absence of PAP, oxidation following reduction of PST was required to completely interconvert the two forms of PST. According to the experimental results, a mechanism for the formation of the two enzyme forms was proposed.
磺基转移酶催化核苷酸与多种亲核试剂之间的硫酰基转移，这些亲核试剂可以是糖类、蛋白质、外源性物质和其他小分子。在酶催化的硫酰基转移反应中，核苷酸可以作为共底物、辅因子、抑制剂或调节剂。我们正在试图了解核苷酸如何通过两种酶形式的表达来调节酚磺基转移酶（PST）的活性。从大肠杆菌中获得了均一的大鼠重组PST，并对共纯化的核苷酸进行了检测。在高盐和氧化条件下，核苷酸从无活性的PST中完全去除。在还原环境中孵育后，总酶活性得以恢复。已知许多核苷酸与PST紧密结合，但通过离子对反相高效液相色谱、紫外可见光谱、³¹P核磁共振以及电喷雾电离质谱和串联质谱分析，仅鉴定出一种核苷酸，即3'-磷酸腺苷5'-磷酸（PAP）与PST结合。除了PAP的存在与否外，PST还原后的氧化对于两种PST形式的完全相互转化是必需的。根据实验结果，提出了两种酶形式形成的机制。
