Determination of substrate specificity of carboxylases by nuclear magnetic resonance.
Determination of substrate specificity of carboxylases by nuclear magnetic resonance.
复制标题
通过核磁共振测定羧化酶的底物特异性。
DOI：
10.1016/0003-2697(87)90404-0
复制
发表时间：
1987
影响因子：
2.9
通讯作者：
J. Hermes
中科院分区：
文献类型：
--
作者：
M. O'Leary;J. Hermes
确定CO2或HCO 3 −是否是酶促羧化的底物通常是利用这两种化合物在低于15°C的温度下需要一分钟以上的平衡这一事实来完成的;因此，根据CO2或HCO 3 −是否用于引发反应，可以获得不同的羧化动力学。本文报道了一种以13 C18 O2为底物测定羧化酶CO2 HCO 3 −专一性的新方法。如果CO2是底物，那么含13 C的产物的18 O含量与使用的13 CO2相同，而如果HCO 3-是底物，则18 O含量是起始原料的2 3。该方法不依赖于CO2 HCO 3 −相互转化的详细动力学，也不依赖于污染的未标记CO2或HCO 3 −的存在。同位素分析采用~（13）C NMR。该方法已被用于确认HCO 3 −是磷酸烯醇式丙酮酸羧化酶的底物。磷NMR谱中氧-18同位素位移的研究证实了在磷酸烯醇丙酮酸的羧化过程中标记物从HC 18 O3-转移到Pidure中的观察结果。
Determination of whether CO2or HCO3−is the substrate for an enzymatic carboxylation has generally been accomplished by taking advantage of the fact that equilibration of these two compounds requires more than a minute at temperatures below 15°C; thus different kinetics of carboxylation are obtained depending on whether CO2or HCO3−is used to initiate the reaction. We report a new method using13C18O2as substrate for determining the CO2HCO3−specificity of carboxylases. If CO2is the substrate, then the18O content of the13C-containing product is the same as that of the13CO2used, whereas if HCO3−is the substrate, the18O content is 2 3 that of the starting material. The method is independent of the detailed kinetics of the CO2HCO3−interconversion and independent of the presence of contaminating unlabeled CO2or HCO3−. Isotopic analysis is accomplished by13C NMR. The method has been used to confirm that HCO3−is the substrate for phosphoenolpyruvate carboxylase. Studies of oxygen-18 isotope shifts in phosphorus NMR spectra have permitted confirmation of the observation that label is transferred from HC18O3−into Piduring the carboxylation of phosphoenolpyruvate.