D-Phe complexes of zinc and cobalt carboxypeptidase A

The binding of D-phenylalanine, D-Phe, to both zinc and cobalt carboxypeptidase A, ZnCPD and CoCPD, has been investigated by a combination of kinetic and spectroscopic techniques. Kinetic studies of the ZnCPD catalyzed hydrolysis of dansyl-Gly-Ala-L-Phe indicate that D-Phe inhibition occurs through a two-site sequential competitive inhibition mode with Ki values of 45 microM and 11.6 mM at pH 8.4, 1 M NaCl, 25 degrees C. Spectral titration of CoCPD under the same conditions indicates a very strong binding mode of D-Phe (KD < 100 microM) that only slightly perturbs the visible cobalt electronic transitions. However, the conversion of CoCPD.D-Phe into a CoCPD.D-Phe2 (KD, 1.13 mM) is accompanied by a very strong spectral perturbation resulting in a complex that is characterized by Amax values of 506 nm (epsilon = 27 M-1 cm-1) and 605 nm (epsilon = 17 M-1 cm-1) and a shoulder at 530 nm (epsilon = 23 M-1 cm-1). The spectral properties of this ternary complex differ markedly from that of the CoCPD.L-Phe.N3-ternary complex. X-ray absorption fine structure, XAFS, studies indicate that these differences are likely due to a more regular tetrahedral coordination sphere for the ternary azide complexes compared to an octahedral coordination geometry for the Zn and CoCPD.D-Phe2 complexes.