Schulte D, Hengstenberg W
Arbeitsgruppe Physiologie der Mikroorganismen, Department of Biology, Ruhr-Universität Bochum, D- 44780 Bochum, Germany.
Protein Eng. 2000 Jul;13(7):515-8. doi: 10.1093/protein/13.7.515.
Several amino acids in the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis were replaced by the corresponding residues in homologous enzymes of glycosidase family 1 with different specificities. Three mutants, W429A, K435V/Y437F and S428D/ K435V/Y437F, were constructed. W429A was found to have an improved specificity for glucosides compared with the wild-type, consistent with the theory that the amino acid at this position is relevant for the distinction between galactosides and glucosides. The k(cat)/K(m) for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-beta-D-galactose-6-phosphate which is the preferred substrate of the wild-type enzyme. This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group. The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosphorylated substrates. The triple mutant is inactive. The double mutant has a dramatically decreased ability to bind o-nitrophenyl-beta-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-beta-D-galactose is barely altered. This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.
乳酸乳球菌6-磷酸-β-半乳糖苷酶活性中心的几个氨基酸被糖苷酶家族1中具有不同特异性的同源酶的相应残基所取代。构建了三个突变体，即W429A、K435V/Y437F和S428D/K435V/Y437F。与野生型相比，发现W429A对葡萄糖苷具有更高的特异性，这与该位置的氨基酸与区分半乳糖苷和葡萄糖苷相关的理论一致。对邻硝基苯基-β-D-葡萄糖-6-磷酸的k(cat)/K(m)比对邻硝基苯基-β-D-半乳糖-6-磷酸（野生型酶的首选底物）高8倍。这表明在突变体中，活性位点残基（可能是Gln19或Trp421）与C-4羟基之间形成了新的氢键。测试了另外两个在磷酸结合环中有交换的突变体结合磷酸化底物的能力。三重突变体无活性。双重突变体结合邻硝基苯基-β-D-半乳糖-6-磷酸的能力显著下降，而与邻硝基苯基-β-D-半乳糖的相互作用几乎没有改变。该结果表明，尽管活性位点的结构高度保守，但来自白车轴草的6-磷酸-β-半乳糖苷酶和相关的生氰β-葡萄糖苷酶对底物具有不同的识别机制。
