HNMR of succinate binding to aspartate transcarbamylase. A comparison of results in D2O and H2O.

The interaction of succinate with asparatete transcarbamylase from Escherichia coli has been studied by magnetic resonance relaxation measurements of the dicarboxylic acid methylene protons in H2O solutions. The pH and temperature dependence of the relaxation in the presence of either native asparte transcarbamylase or its catalytic subunit in H2O solutions is qualitatively very similar to the corresponding situation utilizing D2O as the solvent. From previous result of measurements in D2O[C.B. Beard and P.G. Schmidt, Biochemistry 12(1973)2255] a mechanism was proposed involving 2 protonated groups affecting succinate binding and titratable over the pH range 7-10. Quantitatively, fitting the data from H2O solutions to the mechanism yeilds values of the fitting parameters generally in good agreement with the D2O experiments. The main exceptions are the pKa values calculated for the two titratable groups. For these species the values obtained in the presence of the catalytic subunit are 6.7 and 7.8 in H2O solutions versus 7.3 and 8.6 in D2O solutions. In the presence of native enzyme the corresponding values are 6.8 and 8.3 in H2O versus 7.6 and 9.2 in D2O. These observed differences are consistent with differences in ionization constants of weak acids in D2O relative to H2O. The results imply that succinate interaction with the enzyme active site is similar in the two solvents.