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 Ni^II⋯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.