Schönbrunn E, Svergun D I, Amrhein N, Koch M H
Institute of Plant Sciences, Swiss Federal Institute of Technology, Zurich.
Eur J Biochem. 1998 Apr 15;253(2):406-12. doi: 10.1046/j.1432-1327.1998.2530406.x.
The enzyme UDP-N-acetylglucosamine (UDP-GlcNAc) enolpyruvyltransferase (MurA), the target of the antibiotic fosfomycin, was investigated by small-angle X-ray scattering (SAXS) and fluorescence spectroscopy to detect conformational changes that had been proposed on the basis of the crystal structure of unliganded and liganded MurA. The SAXS data indicate that binding of UDP-GlcNAc to free enzyme results in substantial conformational changes, which can be interpreted as the transition from an open to a closed form. Fosfomycin did not affect the structure of free enzyme or sugar-nucleotide-bound MurA. Phosphoenolpyruvate (pyruvate-P) appeared to induce a structural change upon addition to free enzyme, which differed from that observed upon binding of UDP-GlcNAc. Fluorescence experiments were performed using the hydrophobic fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS). The fluorescence quenching of MurA/ANS solutions upon addition of UDP-GlcNAc or pyruvate-P was concentration dependent in a saturating manner, yielding apparent dissociation constants of K(d(UDP-GlCNAc)) = 59 microM and K(d(pyruvate-P)) = 240 microM. The results suggest that binding of substrates does not exclusively follow an ordered mechanism with UDP-GlcNAc binding first, although binding of UDP-GlcNAc to free enzyme is preferred and possibly influenced by pyruvate-P. The reaction thus appears to follow an induced-fit mechanism, in which the binding site for fosfomycin, and presumably also for pyruvate-P, is created by the interaction of free enzyme with the sugar nucleotide. The methods described here provide a tool for the characterization of site-directed mutants of MurA and the interaction of this enzyme with potential inhibitors.
抗生素磷霉素的作用靶点——酶UDP-N-乙酰葡糖胺（UDP-GlcNAc）烯醇丙酮酸转移酶（MurA），通过小角X射线散射（SAXS）和荧光光谱法进行了研究，以检测基于未结合配体和结合配体的MurA晶体结构所提出的构象变化。SAXS数据表明，UDP-GlcNAc与游离酶的结合导致了显著的构象变化，这可以解释为从开放形式到封闭形式的转变。磷霉素不影响游离酶或糖核苷酸结合的MurA的结构。磷酸烯醇丙酮酸（丙酮酸-P）添加到游离酶后似乎会诱导结构变化，这与UDP-GlcNAc结合时观察到的变化不同。使用疏水荧光探针8-苯胺基-1-萘磺酸盐（ANS）进行了荧光实验。添加UDP-GlcNAc或丙酮酸-P后，MurA/ANS溶液的荧光猝灭呈浓度依赖性饱和，UDP-GlcNAc的表观解离常数K(d(UDP-GlCNAc)) = 59 μM，丙酮酸-P的表观解离常数K(d(pyruvate-P)) = 240 μM。结果表明，底物的结合并不完全遵循UDP-GlcNAc先结合的有序机制，尽管UDP-GlcNAc与游离酶的结合是优先的，并且可能受丙酮酸-P的影响。因此，该反应似乎遵循诱导契合机制，其中磷霉素以及可能还有丙酮酸-P的结合位点是由游离酶与糖核苷酸的相互作用产生的。这里描述的方法为表征MurA的定点突变体以及该酶与潜在抑制剂的相互作用提供了一种工具。
