Balachandran Naresh, To Frederick, Berti Paul J
Department of Chemistry & Chemical Biology and ‡Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Biochemistry. 2017 Jan 31;56(4):592-601. doi: 10.1021/acs.biochem.6b01211. Epub 2017 Jan 13.
3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with K = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KKK/k) versus log(K), revealed a slope, m, of 0.34, with r = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.
3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸（DAHP）合酶在莽草酸生物合成途径的第一步中催化磷酸烯醇丙酮酸（PEP）与赤藓糖-4-磷酸（E4P）发生类似羟醛的反应，生成DAHP。DAHP肟（其中肟取代了酮）是一种强效抑制剂，其K值为1.5 μM。使用DAHP合酶突变体对DAHP肟抑制进行的线性自由能关系（LFER）分析表明，过渡态稳定化与抑制之间存在极好的相关性。重新推导了LFER分析的方程，以规范过渡态稳定化自由能和抑制剂结合自由能按比例而非相等变化的可能性，这与文献中大多数LFER分析显示非单位斜率的事实一致。斜率为1（m = 1）表明催化和抑制剂结合对诸如突变或修饰的抑制剂/底物结构等扰动同样敏感。斜率<1或>1分别表明抑制剂结合对扰动的敏感性低于或高于催化。使用四分子特异性常数进行LFER分析，即绘制log(KKK/k)对log(K)的图，得到斜率m为0.34，r为0.93。这提供了证据表明DAHP肟正在模拟DAHP合酶反应的第一个不可逆过渡态，推测是磷酸从四面体中间体离去。这证明肟基团可以作为磷酸基团的功能以及结构模拟物。
