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  • Title: Determination of substrate specificity of carboxylases by nuclear magnetic resonance.
    Author: O'Leary MH, Hermes JD.
    Journal: Anal Biochem; 1987 May 01; 162(2):358-62. PubMed ID: 3111298.
    Abstract:
    Determination of whether CO2 or 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 degrees C; thus different kinetics of carboxylation are obtained depending on whether CO2 or HCO3- is used to initiate the reaction. We report a new method using 13C18O2 as substrate for determining the CO2/HCO3- specificity of carboxylases. If CO2 is the substrate, then the 18O content of the 13C-containing product is the same as that of the 13CO2 used, whereas if HCO3- is the substrate, the 18O content is 2/3 that of the starting material. The method is independent of the detailed kinetics of the CO2/HCO3- interconversion and independent of the presence of contaminating unlabeled CO2 or HCO3-. Isotopic analysis is accomplished by 13C 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 Pi during the carboxylation of phosphoenolpyruvate.
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