The Gene Structure of Starch Phosphorylase from Sweet Potato
The Gene Structure of Starch Phosphorylase from Sweet Potato
复制标题
甘薯淀粉磷酸化酶的基因结构
作者：
C. T. Lin;M. Lin;H. Chou;P. D. Lee;J. Su
在微生物、动物和植物中，a-葡聚糖磷酸化酶是控制储存多糖进入糖酵解途径的关键酶。这些酶催化葡聚糖底物中cu- 1,4 -糖苷键的可逆磷酸解。在高等植物中，以马铃薯为研究对象最多。根据对支链葡聚糖的亲和度高低，将其分为L型和H型（Nakano et al., 1989）。L型比H型更大，因为在中心位置插入了78个氨基酸残基，导致该分子与其他来源的磷酸化酶的同源性较低（Nakano and Fukui, 1986; Nakano et al., 1989; Lin et al., 1991; Mori et al., 1991）。额外肽的存在被认为会影响底物亲和力（Nakano and Fukui, 1986），并且通过蛋白质工程方法证实了这一点（Mori et al., 1993）。动物糖原磷酸化酶具有共价和变构两种调节机制（Fukui et al., 1982; Hwang and Fletterick, 1986），它们的结构-功能关系已经建立，但对于任何一种Lor h型植物磷酸化酶，都不知道这种功能和相应的结构。以前，我们假设生长中的甘薯（Ipomoea batatus L.）根中的SP可能参与淀粉合成途径。甘薯SP凝胶电泳分析呈现多条带，表明在中链78-残基段存在不同蛋白水解敏感性的亚基。氨基酸序列的测定受到N端阻断和难以获得完整的纯肽的阻碍。我们已经克隆并测序了编码这种l型同工酶的cDNA （Lin et al., 1991）。该cDNA编码一个955个残基的多肽，与l型马铃薯酶有81%的同源性（916个残基加上一个假定的50个残基的中转肽）。这两种酶在N端大约70个残基（包括一个假定的传递肽）和中链78个残基插入中发现了较高的差异。马铃薯之间的相似性非常高
a-Glucan phosphorylases are of key importance among the enzymes controlling the entry of storage polysaccharides into the glycolytic pathway in microbes, animals, and plants. The enzymes catalyze the reversible phosphorolysis of cu-l,4-glucosidic linkages in glucan substrates. Among SP from higher plants, those from potato are best studied. They are classified into L and H types depending on their low and high affinities toward branched glucans, respectively (Nakano et al., 1989). The L type is larger than the H type in having a 78-amino acid residue insertion at the central position, causing the molecule to show a low homology toward phosphorylases from other sources (Nakano and Fukui, 1986; Nakano et al., 1989; Lin et al., 1991; Mori et al., 1991). The presence of the extra peptide was suggested to affect the substrate affinity (Nakano and Fukui, 1986), and this was proven to be true by a protein engineering approach (Mori et al., 1993). Animal glycogen phosphorylases are endowed with both covalent and allosteric regulatory mechanisms (Fukui et al., 1982; Hwang and Fletterick, 1986) and their structurefunction relationship has been established, but neither such functions nor the corresponding structures are known for either Lor H-type plant phosphorylases. Previously, we postulated that SP in the growing sweet potato (Ipomoea batatus L.) root could be involved in a starch synthesis pathway. Gel electrophoresis analysis of sweet potato SP always gave multiple bands, which indicated the presence of subunits with different proteolytic susceptibility at the midchain 78-residue segment. Determination of amino acid sequence was hindered by blocking of the N terminus and the difficulty of obtaining intact pure peptide. We have cloned and sequenced a cDNA encoding this L-type isozyme (Lin et al., 1991). The cDNA encodes a 955-residue polypeptide that has 81 % homology toward the L-type potato enzyme (916 residues plus a 50-residue putative transit peptide). Higher divergence of the two enzymes was found in about 70 residues of the N termini, including a putative transit peptide, and in the midchain 78-residue insert. Very high similarity between the potato