Change of lactate dehydrogenase function by protein engineering

在戊糖乳杆菌D-LDH中，Phe 299的取代导致酶对2-酮酸底物的亲和力和催化速率显著降低，并改变了酶反应的一级同位素效应，表明Phe 299在刺激2-酮酸底物结合和随后的催化反应中起重要作用。特别是取代显着破坏丙酮酸还原反应中的2-酮酸，表明Phe 299在特异性识别底物C3部分也起着重要作用。Ser 102的取代诱导了底物结合的显著正协同性，表明底物结合位点的结构与整个蛋白质结构协同改变。另一方面，在干酪乳杆菌变构L-LDH中，调节位点中His 205的替换诱导丙酮酸的同向调节的极大损失，从而将酶改变为绝对果糖1，6-二磷酸依赖性酶。通过酶动力学和化学修饰的分析强烈地表明，His 205不是以非活性状态参与丙酮酸结合，而是以酶结构的活性状态参与丙酮酸结合。因此，His 205可能通过与丙酮酸的相互作用引发变构转换。戊糖乳杆菌非变构型L-LDH与干酪乳杆菌L-LDH具有高度的序列相似性，通过晶体衍射分析可以建立其三维结构。此外，为了确定L.casei L-LDH的三维结构，通过对最佳结晶条件的筛选，获得了最大粒径为1.25 mm的晶体。

ln Lactobocillus pentosus D-LDH,replacements of Phe299 induced marked reductions in the affinity to 2-ketoacid substrates and the catalytic velocity, and also changed primary isotope effects on the enzyme reaction, indicating that Phe299 plays essential roles to stimulate the binding of 2-ketoacid substrates and subsequent catalytic reaction. The replacements Particularly markedly damaged the pyruvate reduction among the reactions for 2-ketoacids, indicating that Phe299 plays also important role in the specific recognition of the substrate C3 moiety. Replacement of Serl02 induced a significant positive cooperativity on the substrate binding, indicating that the structure of the substrate binding site is coordinately changed with the whole protein structure. In L.casei allosteric L-LDH,on the other hand, replacement of His205 in the regulatory site induced a great loss of the homotropic regulation by pyruvate, and thereby change the enzyme to a absolutely fructose 1,6-bisphosphate-dependent enzyme. Analysis by the enzyme kinetics and chemical modification strongly suggested that His205 is involved in the pyruvate binding not in an inactive state, but in an active state of the enzyme structure. Therefore, His205 possibly triggered the allosteric transition through the interactions with pyruvate. In L.pentosus non-allosteric L-LDH,which shows high sequence similarity with L.casei L-LDH,the three-dimensional structure could be built through its crystarographic analysis. To also determine three dimensional structure of L.casei L-LDH,in addition, the crystals of the enzyme, the maximal size of which was I.25 mm, were obtained by screening optimal conditions for crystallization.