Abdelghany H M, Gasmi L, Cartwright J L, Bailey S, Rafferty J B, McLennan A G
School of Biological Sciences, University of Liverpool, UK.
Biochim Biophys Acta. 2001 Nov 26;1550(1):27-36. doi: 10.1016/s0167-4838(01)00263-1.
Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.
在成年秀丽隐杆线虫提取物中检测到不对称切割5',5"'-P(1),P(4)-四磷酸二腺苷（Ap4A）的水解酶活性，并在大肠杆菌中扩增并表达了相应的cDNA。正如预期的那样，序列分析表明该酶是Nudix水解酶家族的成员。纯化的重组酶表现为典型的动物Ap4A水解酶。它以7微摩尔的K(m)和27秒(-1)的k(cat)水解Ap4A，产生AMP和ATP作为产物。它对其他具有四个或更多磷酸基团的腺苷和二腺苷多磷酸也有活性，但对二腺苷三磷酸没有活性，总是产生ATP作为产物之一。它受到氟化物的非竞争性抑制（K(i)=25微摩尔），以及以Ap4A为底物时受到腺苷-5'-四磷酸的竞争性抑制（K(i)=10纳摩尔）。很容易获得具有矩形板形态的高质量衍射晶体并收集了初步数据。这些晶体的最小d间距衍射到2埃，属于空间群C222或C222(1)。对Nudix家族已知和推定的Ap4A水解酶的系统发育分析表明，它们分为两组，一组包括植物和变形菌门的酶，另一组包括动物和古菌的酶。秀丽隐杆线虫Ap4A水解酶的完整结构测定将有助于确定这种分组的基础。
