Conversion of bacterial allosteric L-lactate dehydrogenases to constitutively active enzymes

对于LCLDH， S67E、N68D、E178K和A235K替代物增加了与fbp无关的活性，降低了底物Km值。x射线晶体学表明，这些突变在q轴相关亚基之间引入了亚基间盐桥。对于LPLDH， D68N和D68H的替换一致地将双曲底物饱和曲线改变为s型曲线。在TCLDH的情况下，L67E、N68D、E178K和A235K（或A235R）替换（Q突变）在没有FBP的情况下仅略微提高了底物Km，尽管它们提高了Vm值，相反，R173Q和R216L替换（P突变）显著提高了底物Km。p轴和q轴突变增加了与fbp无关的活性和热稳定性。结构比较表明，LCLDH和TCLDH的结构变化差异很大，因此需要不同的设计来引入高的本构催化活性。

For LCLDH, S67E, N68D, E178K and A235K replacements additively increased the FBP-independent activity, reducing the substrate Km values. The X-ray crystallography indicated that these mutations introduced the inter-subunit salt bridges between the Q-axis related subunits. For LPLDH, D68N and D68H replacements consistently changed the hyperbolic substrate saturation curves to the sigmoidal ones. In the case of TCLDH, L67E, N68D, E178K and A235K (or A235R) replacements (Q mutations) only slightly improved substrate Km in the absence of FBP, though they improved Vm values, instead R173Q and R216L replacements (P mutations) markedly improved the substrate Km. The P-axis and Q-axis mutations additively improved the FBP-independent activity and thermal stability. Structural comparison indicated that LCLDH and TCLDH exhibit greatly different structural changes, and therefore require the different designs that introduce the constitutively high catalytic activities.