X-ray absorption fine structure study of the active site of zinc and cobalt carboxypeptidase A in their solution and crystalline forms

• Published in: Biochemistry (Easton) 1992-02, Vol.31 (4), p.1159-1168
• Main Authors: Zhang, Ke, Chance, Britton, Auld, David S, Larsen, Kjeld S, Vallee, Bert L
• Format: Article
• Language: English
• Subjects: active sites; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; carboxypeptidase A; Carboxypeptidases - chemistry; Carboxypeptidases - genetics; Carboxypeptidases A; Catalysis; Cattle; Cobalt - chemistry; crystal structure; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Models, Molecular; Mutagenesis; Particle Accelerators; Solutions; Spectrometry, X-Ray Emission; X-ray crystallography; X-Ray Diffraction; Zinc - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00119a027

A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of atoms, with four atoms (N or O) located at an average distance of 2.03 +/- 0.01 A and one atom (N or O) located at 2.57 +/- 0.04 A. The four-atom distribution remains the same for ZnCPD in its crystalline state, but the fifth atom is found at 2.36 +/- 0.04 A. Examination of the higher coordination shell, between 2.7 and 4.2 A, reveals the presence of two imidazoles. Combined with X-ray crystallographic results, a structural model is proposed. The four atoms at an average distance of 2.03 A are assigned to the two delta 1 nitrogens of His-69 and His-196, one epsilon 1 oxygen of Glu-72, and the oxygen of a coordinated water molecule. The atom at 2.57 A for ZnCPD in solution is assigned to the epsilon 2 oxygen of Glu-72. The results for CoCPD in solution are similar with the four atoms at an average distance of 2.08 +/- 0.01 A and one atom at 2.50 +/- 0.04 A, which moves to 2.34 +/- 0.04 A in the crystalline enzyme. The intensity of the 3d "pip" peak for CoCPD is consistent with a distorted tetragonal metal geometry for the solution form of the enzyme which is converted to a more pentacoordinated metal site for the crystalline enzyme. The first shell distribution of crystalline CoCPD is quite disordered, which may be largely due to the disorder of His-69 and His-196 as indicated by higher shell analysis. Thus, the XAFS studies show that the metal coordination spheres in the zinc and cobalt enzymes are quite similar in the solution state but differ from their crystalline counterparts. The XAFS studies provide the necessary background for measurement of substrate- and inhibitor-promoted structural changes in the metal coordination sphere of the zinc and other metal-substituted carboxypeptidases in the solution state.