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.
European Molecular Biology Laboratory, Grenoble Outstation, France.
K S
KS
Larsen
K
K
Zhang
D S
DS
Auld
D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002417:Cattle; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D010649:Phenylalanine / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D017550:Spectroscopy, Fourier Transform Infrared; D015032:Zinc / Q000378:metabolism*
D003035:Cobalt; D010649:Phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc