Determination of substrate specificity of carboxylases by nuclear magnetic resonance.
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.
D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010103:Oxygen Isotopes; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D013379:Substrate Specificity
D001639:Bicarbonates; D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D002245:Carbon Dioxide; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase