Nuclear magnetic resonance study of ligand binding to Mn-aspartate transcarbamylase

Aspartate transcarbamylase from Escherichia coli has been prepared with up to four of zinc ions replaced by manganese, and the effect of this substitution on the proton nuclear magnetic resonance properties of succinate bound to the catalytic site and of cytidine 5'-triphosphate bound to the regulatory site has been determined, The specific activity and allosteric properties of the Mn-substituted enzyme are essentially identical with those of the native enzyme. The longitudinal relaxation time, T1, of the succinate protons is shortened by the native enzyme and is shortened further by the Mn-substituted enzyme at both 100 and 220 MHz in D2O solutions of 0.02 M immidazole chloride (pH 7.0), 10 minus 3 M beta-mercaptoethanol, 0;2 mM ethylenediamenetetraacetic acid, and 2.5 mM carbamyl phosphate over a temperature range of 5 to 35 degrees. Under the same conditions, the transverse relaxation time, T2, of the succinate protons at 90 MHz is shortened to the same extent by native and Mn-substituted enzyme. The temperature dependence of the relaxation times indicates that the shortening of the transverse relaxation time is determened by the lifetime of bound succinate, whereas the further shortening of the longitudinal relaxation time by the Mn-substituted enzyme is due to dipolar relaxation, i.e. to the interaction between Mn and the succinate protons. The distance between the Mn and the protons of succinate bound to the enzyme can be calculated from the relaxation time measurements and is 15,3 A. The dipolar interaction correlation time which is needed for the calculation of this distance, was found to be 3.5 X 10 minus 9 sec from the frequency dependence of T1. The transverse relaxation time of the C-6 proton of CTP is shortened to the same extent by both the native and Mn-substituted enzyme in D2O solutions of 0.02 M imidazole chloride (pH 7.0), 10 MINUS 3 M beta-mercaptoethanol, 0.2 mM ethylenediaminetetraacetic acid, and 2.5 mM carbamyl phosphate over the temperature 5-30 degrees. Since the temperature depencece of the relaxation time indicates the relaxation is not exchange limited, the manganese must be too distant from the bound CTP for an appreciable interaction to occur. This requires that the manganese be greater than 20A from the CTP. These results are used together with other available structural data to construct a schematic model for aspartate transcarbamylase.