Balachandran Naresh, Heimhalt Maren, Liuni Peter, To Frederick, Wilson Derek J, Junop Murray S, Berti Paul J
Department of Chemistry, York University , Toronto, Ontario M3J 1P3, Canada.
Biochemistry. 2016 Dec 6;55(48):6617-6629. doi: 10.1021/acs.biochem.6b00930. Epub 2016 Nov 17.
3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with K = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, t, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The k value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent K value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.
3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸（DAHP）合酶催化莽草酸途径的第一步。它催化磷酸烯醇丙酮酸（PEP）与赤藓糖-4-磷酸（E4P）发生类似羟醛缩合的反应，生成DAHP。其动力学机制为快速平衡有序的双双机制，必需的二价金属离子锰首先结合，随后是PEP和E4P。DAHP肟中，肟基取代了酮氧，是一种强效抑制剂，K = 1.5±0.4 μM，不过在高抑制剂浓度下仍有残余活性。它表现出慢结合抑制，停留时间t为83分钟。晶体结构显示，肟官能团与两个结晶水结合在催化中心的同一位置，该位置与四面体中间体的磷酸基团相同。DAHP合酶具有二聚体的二聚体同四聚体结构，DAHP肟仅与每个紧密二聚体的一个亚基结合。抑制剂结合在其结合的亚基中对所有三种底物具有竞争性。DAHP肟不与金属结合位点重叠，因此它们相互排斥结合的原因尚不清楚。同样，抑制剂仅在两个亚基中结合也没有明显的结构原因；然而，整体氢/氘交换的变化表明，抑制剂结合后蛋白质动力学发生了大规模变化。高抑制剂浓度下残余活性的k值降低了3倍，表观K值至少降低了10倍。亚基B和C中的DAHP肟与亚基A和D中的E4P之间结合的这种正协同性似乎是高抑制剂浓度下抑制不完全的主要原因。尽管肟与磷酸盐缺乏明显的结构相似性，但肟和结晶水起到了一种小的中性磷酸盐模拟物的作用。
