D-Phe complexes of zinc and cobalt carboxypeptidase A

• Published in: Journal of inorganic biochemistry Vol. 64; no. 3; pp. 149 - 162
• Main Authors: Larsen, Kjeld S., Zhang, Ke, Auld, David S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-11-1996
• Subjects: 3-([ tris(hydroxyethyl)methyl]-amino) propanesulfonic acid; 3-(N-morpholino)propanesulfonic acid; acetate; Animals; Carboxypeptidases - metabolism; Carboxypeptidases A; Cattle; cobalt (II)-substituted carboxypeptidase A; Cobalt - metabolism; CoCPD; Hepes; imidazole; Kinetics; Mops; N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; native carboxypeptidase A; Phenylalanine - metabolism; radial distribution function; RDF; Spectrophotometry, Atomic; Spectroscopy, Fourier Transform Infrared; Taps; Zinc - metabolism; ZnCPD; Mops; Ac; ZnCPD; Im; RDF; Hepes; Taps; CoCPD
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/0162-0134(96)00037-2

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 K
i values of 45 μM and 11.6 mM at pH 8.4, 1 M NaCl, 25°C. Spectral titration of CoCPD under the same conditions indicates a very strong binding mode of D-Phe (K
D < 100 μM) that only slightly perturbs the visible cobalt electronic transitions. However, the conversion of CoCPD · D-Phe into a CoCPD · D-Phe
2 (K
D, 1.13 mM) is accompanied by a very strong spectral perturbation resulting in a complex that is characterized by λ
max values of 506 nm (ϵ = 27 M
−1 cm
−1) and 605 nm (ϵ = 17 M
−1 cm
−1) and a shoulder at 530 nm (ϵ = 23 M
−1 cm
−1). The spectral properties of this tenary complex differ markedly from that of the CoCPD · L-Phe · N
3
− 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-Phe
2 complexes.