Nuclear magnetic resonance study of ligand binding to manganese-aspartate transcarbamylase (original) (raw)

Aspartate transcarbamylase from Escherichia coli has been prepared with up to four of the 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 Mnsubstituted enzyme are essentially identical with those of the native enzyme. The longitudinal relaxation time, T I , of the succinate protons is shortened by the native enzyme and is shortened further by the Mn-substituted enzyme a t both 100 and 220 M H z in DzO solutions of 0.02 M imidazole chloride (pH 7.0), M 6-mercaptoethanol, 0.2 m M ethylenediaminetetraacetic acid, and 2.5 m M carbamyl phosphate over a temperature range of 5 to 35O. Under the same conditions, the transverse relaxation time, T2, of the succinate protons at 90 M H z 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 determined by the lifetime of bound succinate, whereas the further shortening A s p a r t a t e transcarbamylase is an allosteric enzyme regulated by nucleotides. This enzyme has been extensively studied with equilibrium, kinetic, and structural techniques (cf. Jacobson and Stark, 1973; Hammes and Wu, 1974). The native enzyme contains two trimeric catalytic subunits and three dimeric regulatory subunits (Gerhart and