A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases
A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases
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DOI：
10.1093/jb/mvr100
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发表时间：
2011-11-01
影响因子：
2.7
通讯作者：
Taguchi, Hayao
中科院分区：
文献类型：
--
作者：
Arai, Kazuhito;Ichikawa, Jun;Taguchi, Hayao
干酪乳杆菌L-乳酸脱氢酶(L-乳酸脱氢酶)是一种典型的细菌变构L-乳酸脱氢酶，其酶活性需要1，6-二磷酸果糖(FBP)。设计了一个突变体，模仿戊糖乳杆菌非变构L-乳酸脱氢酶(LPLDH)，在Q轴亚单位界面引入了一个亚基间盐桥网络。突变的LCLDH具有较高的催化活性，其丙酮酸饱和曲线与FBP无关，在pH 5.0时的K-m和V-m值与含有FBP的完全激活的野生型酶相当，但在pH 7.0时，FBP或Mn2+使K-m值略有改善。在有FBP存在的情况下，突变酶的表观变性温度(T-1/2)明显高于野生型酶，而在没有FBP的情况下，突变酶的表观变性温度(T-1/2)更低，表现出更高的失活活化热(Delta H-双匕首)。这一结果与LCLDH变构平衡中活化态比非活化态更不稳定的事实是一致的。在许多细菌非变构L-乳酸脱氢酶和二聚体L-苹果酸脱氢酶中，LPLDH样网络似乎是保守的，因此是细菌L-乳酸脱氢酶在进化过程中功能分化的关键。
l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K-m and V-m values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K-m value was slightly improved with FBP or Mn2+ at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T-1/2) than the wild-type enzyme in the presence of FBP, but showed an even lower T-1/2 without FBP, where it exhibited higher activation enthalpy of inactivation (delta H-double dagger). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.