Nakai T, Konishi T, Zhang X Q, Chollet R, Tonouchi N, Tsuchida T, Yoshinaga F, Mori H, Sakai F, Hayashi T
Wood Research Institute, Kyoto University, Japan.
Plant Cell Physiol. 1998 Dec;39(12):1337-41. doi: 10.1093/oxfordjournals.pcp.a029339.
A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.
通过对大肠杆菌中表达的重组蛋白进行定点诱变，对绿豆（Vigna radiata Wilczek）蔗糖合酶进行了突变分析，其中在Ser11处引入了两个不同的酸性氨基酸残基（Asp或Glu）（S11D、S11E）。只有野生型酶（Ser11）在体外被来自大豆根瘤的钙依赖性蛋白激酶磷酸化，这表明这是绿豆蔗糖合酶中的特定靶残基。这种磷酸化酶以及S11D和S11E突变酶对蔗糖的表观亲和力增加，尽管野生型、磷酸化野生型和突变酶对UDP-葡萄糖和果糖的亲和力相似。这些结果表明，11位的单阴离子（1-）侧链模拟Ser11-P2-残基以结合和裂解蔗糖用于UDP-葡萄糖的合成。由于S11E突变酶显示出最低的K（m）（蔗糖）和重组蛋白的最高催化效率，因此对该S11E突变体的酶学性质进行了进一步表征。结果表明，用Glu11取代Ser11适度保护了蔗糖合成活性免受酚糖苷的影响，并改变了酶的核苷酸特异性。我们推测，在Ser11处引入负电荷可能参与了蔗糖合酶的酶促扰动。
