School of Biological Sciences, Queen's University, Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK.
Chembiochem. 2011 Sep 5;12(13):2081-7. doi: 10.1002/cbic.201100308. Epub 2011 Jul 11.
Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.
半乳糖激酶催化半乳糖在 ATP 消耗下的位点和立体特异性磷酸化。通过将一个酪氨酸残基改变为组氨酸，可以拓宽细菌半乳糖激酶酶的特异性。通过测试所有 19 种可能的变体，研究了改变人半乳糖激酶（Tyr379）等效残基的影响。除了 Y379P 之外，所有这些改变都导致在大肠杆菌中表达的可溶性蛋白，并且所有可溶性变体都能够催化半乳糖的磷酸化，除了 Y379A 和 Y379E。变体 Y379C、Y379K、Y379R、Y379S 和 Y379W 都能够催化各种单糖的磷酸化，包括野生型酶不作用的单糖。这些变体半乳糖激酶的新底物包括 D-甘露糖和 D-果糖。后一种单糖被认为以吡喃糖构型反应。分子建模表明，这些改变不会导致酶整体结构的变化。然而，改变 Tyr379 会增加活性位点周围肽骨架的柔韧性。因此，据推测，改变 Tyr379 通过将柔韧性的变化传递到活性位点来影响底物特异性，从而允许更广泛的化合物被容纳。
