Ivanenkov Vasily V, Murphy-Piedmonte Deirdre M, Kirley Terence L
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, P.O. Box 670575, Cincinnati, Ohio 45267-0575, USA.
Biochemistry. 2003 Oct 14;42(40):11726-35. doi: 10.1021/bi035137r.
The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception. NTPDases1-4 are oligomeric integral membrane proteins, whereas NTPDase5 (CD39L4) and NTPDase6 (CD39L2) are soluble monomeric enzymes, making them more amenable to thorough structural and functional analyses than the membrane-bound forms. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6. Consistent with the enzyme expressed in mammalian cells, this recombinant NTPDase6 efficiently hydrolyzes GDP, IDP, and UDP (specific activity of approximately 50000 micromol mg(-1) h(-1)), with slower hydrolysis of CDP, ITP, GTP, CTP, ADP, and UTP and virtually no hydrolysis of ATP. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells. The secondary structure of the refolded enzyme was determined by circular dichroism to be 33% alpha-helix, 18% beta-sheet, and 49% random coil, consistent with the secondary structure predicted from the amino acid sequence of soluble NTPDase6. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not. Mutation of this nonconserved cysteine resulted in an enzyme very similar to wild type in its enzymology and secondary structure, indicating that this cysteine exists as a free sulfhydryl and is not essential for structure or function. The disulfide pairing of the other four cysteine residues was determined as Cys(249)-Cys(280) and Cys(340)-Cys(354) by HPLC and mass spectral analysis of tryptic peptides. Due to conservation of these cysteine residues, these two disulfide bonds are likely to exist in all NTPDases. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed.
胞外核苷三磷酸二磷酸水解酶（NTPDases）可控制细胞外核苷酸浓度，从而调节许多重要的生物学反应，包括血液凝固和痛觉。NTPDases1 - 4是寡聚整合膜蛋白，而NTPDase5（CD39L4）和NTPDase6（CD39L2）是可溶性单体酶，这使得它们比膜结合形式更易于进行全面的结构和功能分析。因此，我们在此报告人NTPDase6可溶性部分的细菌表达、重折叠、纯化及生化特性。与在哺乳动物细胞中表达的酶一致，这种重组NTPDase6能有效水解GDP、IDP和UDP（比活性约为50000微摩尔·毫克⁻¹·小时⁻¹），对CDP、ITP、GTP、CTP、ADP和UTP的水解较慢，对ATP几乎不水解。GDP的米氏常数（K(m)）（130±30微摩尔）与在哺乳动物细胞中表达的可溶性大鼠NTPDase6所测定的相似。通过圆二色性确定重折叠酶的二级结构为33%的α - 螺旋、18%的β - 折叠和49%的无规卷曲，与根据可溶性NTPDase6氨基酸序列预测的二级结构一致。可溶性NTPDase6的五个半胱氨酸残基中有四个在所有NTPDases中高度保守，而第五个残基则不然。这个不保守的半胱氨酸的突变导致一种酶在酶学和二级结构上与野生型非常相似，表明这个半胱氨酸以游离巯基形式存在，对结构或功能并非必需。通过对胰蛋白酶肽段的HPLC和质谱分析，确定其他四个半胱氨酸残基的二硫键配对为Cys(249) - Cys(280)和Cys(340) - Cys(354)。由于这些半胱氨酸残基的保守性，这两个二硫键可能存在于所有NTPDases中。结合这些发现以及其他NTPDases的相关结果，提出了NTPDase6的结构模型。
