Viswanathan T S, Johnson R E, Fisher H F
Biochemistry. 1982 Jan 19;21(2):339-45. doi: 10.1021/bi00531a022.
A study of the various solution forms of alpha-ketoglutaric acid using UV absorption spectrophotometry and 13C NMR spectroscopy shows that at neutral pH alpha-ketoglutarate exists predominantly as the keto form with about 7% hydrated form (gem-diol) and a small amount of cyclic form. Protonation of the gamma-carboxylate group increases the amount of cyclic form to 20% with very little increase in the amount of gem-diol. Protonation of the alpha-carboxylate increases the amount of cyclic form to 30% and the amount of gem-diol to 35%. The pH and temperature dependence of the 13C NMR line widths indicated that the interconversion of keto and cyclic forms is extremely rapid. The rate of interconversion of keto and gem-diol forms was studied spectrophotometrically at various temperatures, buffers, and pH values and by varying the total concentration of alpha-ketoglutarate. The hydration reaction of alpha-ketoglutarate is not catalyzed by bovine liver glutamate dehydrogenase (E) or by the E-NADPH complex. The enzyme uses the keto form of alpha-ketoglutarate as the substrate. The gem-diol form is not itself a substrate but becomes converted to the keto form with a half-life of about 0.3 min at 15 degrees C in 0.1 M potassium phosphate buffer (pH 7.6).
一项运用紫外吸收分光光度法和13C核磁共振波谱法对α-酮戊二酸各种溶液形式的研究表明，在中性pH值条件下，α-酮戊二酸主要以酮式存在，约7%为水合形式（偕二醇），还有少量环状形式。γ-羧基的质子化使环状形式的含量增加到20%，而偕二醇的含量增加极少。α-羧基的质子化使环状形式的含量增加到30%，偕二醇的含量增加到35%。13C核磁共振谱线宽度对pH值和温度的依赖性表明，酮式和环状形式之间的相互转化极其迅速。在不同温度、缓冲液和pH值条件下，通过改变α-酮戊二酸的总浓度，用分光光度法研究了酮式和偕二醇形式之间的相互转化速率。α-酮戊二酸的水合反应不受牛肝谷氨酸脱氢酶（E）或E-NADPH复合物的催化。该酶以α-酮戊二酸的酮式作为底物。偕二醇形式本身不是底物，但在0.1M磷酸钾缓冲液（pH 7.6）中，于15℃时，其半衰期约为0.3分钟，可转化为酮式。
