Spectroscopic studies of the interaction of nickel(II) carboxypeptidase with phosphate and pyrophosphate
Abstract
The interaction between nickel(II)-substituted carboxypeptidase A and the inhibitors phosphate and pyrophosphate has been investigated at pH 7 by electronic absorption and 1H, 31P, and 13C NMR spectroscopies. Upon binding to the nickel enzyme, the longitudinal relaxation rates T1–1 of the 31P nucleus of these inhibitors are enhanced significantly compared to the diamagnetic zinc enzyme. The NiII⋯31P distances indicate that both inhibitors bind directly to the metal ion under rapid-exchange conditions. From the temperature dependence of the isotropic shifts and the molar absorbance values of the 1 : 1 adducts formed, a pseudo-octahedral co-ordination for the metal ion is suggested. Proton NMR titrations of nickel(II) carboxypeptidase with phosphate in the presence and absence of D-phenylalanine (D-Phe) indicate that the occupancy of the S′1 subsite of the enzyme does not substantially modify the affinity of the phosphate anion for the metal ion. Carbon-13 NMR T2–1 measurements on 13CO2-labelled D-Phe show that phosphate or pyrophosphate do not compete for binding to Arg-145. These results are discussed in terms of the general model of anion interaction with carboxypeptidase.