Aspartate transcarbamylase. The use of primary kinetic and solvent deuterium isotope effects to delineate some aspects of the mechanism.
Abstract 14C- and 18O-labeled carbamyl phosphates have been used to study primary kinetic isotope effects in the reaction catalyzed by the catalytic subunit of the aspartate transcarbamylase of Escherichia coli. A novel aspect of the methodology is the use of 14C as a tracer to determine the isotope effect caused by 18O alone, without the need for analysis by mass spectrometry. At optimum concentrations of carbamyl phosphate and l-aspartate and at pH 7.8 (near the pH optimum of the enzyme), the effect of 14C at the carbonyl carbon or 18O at the anhydride oxygen of carbamyl phosphate is very small. One possible explanation, but not the only one, is that kinetic steps in which the bonds to these atoms change are not important in determining the observed rate. Such an interpretation is consistent with a rate-determining conformational change of the enzyme at the pH optimum. At pH 10, where the activity of the enzyme is low, the rate with 14C-labeled carbamyl phosphate is about 95% of the rate with the 12C compound. On the other hand, no substantial effect is seen for 18O in the anhydride position. Taken together, these observations indicate that a step in which the bonding to the carbon atom changes and the bonding to the anhydride oxygen does not change becomes important in the observed rate at pH 10. Such a process might be the formation of a tetrahedral intermediate. The solvent deuterium isotope effect in D2O has also been studied, at optimum concentrations of the substrates. The maximum velocity is essentially the same in D2O and in H2O. Thus, proton transfer does not seem to be involved in a ratedetermining step in the aspartate transcarbamylase reaction. The pH activity profile of the enzyme is shifted up by about 0.8 pH unit in D2O, an effect which can be accounted for approximately quantitatively by the expected change in the pKa of the amino group of l-aspartate.