Analysis of Allosteric mechanism in bacterial L-lactate dehydrogenases

1.突变酶的X射线晶体分析：获得了FBP结合部位的精氨酸-173或组氨酸-188被谷氨酰胺或酪氨酸取代的突变酶。酶的初步分析表明，组氨酸残基是FBP结合所必需的。底物抑制分析：替代实验表明丝氨酸-193和丝氨酸-318在测定米氏常数和抑制常数中起重要作用。晶体的X-射线分析表明，单晶中同时存在T态和R态的酶分子。在2.5埃分辨率下对两种分子结构进行了分析。变构现象的分子机制：FBP在T态的结合克服了P轴上各亚基之间的静电排斥。这诱导了四个亚基之间的旋转，并通过Q轴上亚基之间的相互作用使活性部位活跃。

1. X-ray crystal analysis of mutant enzyme : Mutant enzymes in which arginine-173 or histidine-188 in FBP-binding site were replaced to glutamine or tyrosine were obtained. Preliminary analysis of the enzymes showed that the histidine residue is necessary for the binding of FBP.2. Analysis of substrate inhibition : Replace experiments showed that serine-193 and serine-318 play an important role for determining Michaelis and inhibition constants.3. X-ray analysis of crystal which comprises both T and R forms of the LDH : Crystal analysis revealed that there are enzyme molecules in both T and R states in a single crystal. Both molecular structure were analyzed in 2.5 angstrom resolution.4. Molecular mechanism of allosteric phenomena : Binding of FBP in T state overcomes electrostatic repulsion between subunits in P-axis. This induces rotation among four subunits and makes the active site active by interaction between subunit in Q-axis.