Structure of binary and ternary complexes of zinc and cobalt carboxypeptidase A as determined by X-ray absorption fine structure.
Carboxypeptidase A, ZnCPD, is typical of a wide range of exo- and endo-metalloproteases that have three protein ligands and a water molecule bound to a catalytic zinc atom and a glutamate residue in the active site that likely acts in conjunction with the Zn-bound water to bring about catalysis. Such enzymes generally have bell-shaped pH-activity profiles (EH2 in equilibrium with EH in equilibrium with E) where the concentration of the catalytic species EH is regulated by pKEH2 and pKEH. The present X-ray absorption fine structure, XAFS, study has determined the structure and pH behavior of the binary and ternary product complexes in order to examine the role of the Zn-bound water in catalysis. Increasing the pH from 7 to 10 of the ZnCPD.L-Phe complex results in the same type of progressive spectral changes in the near-edge XAFS spectrum as is seen for ZnCPD, but the changes are complete by more than 1 pH value below that observed for ZnCPD. The results are in agreement with kinetic studies that show E binds the protonated form of L-Phe more tightly than EH, thus in effect decreasing the value of pKEH. The XAFS results show the average interatomic distance. R. for the zinc ligands of the EH.L-Phe complex decreases by 0.02 A upon formation of the E.L-Phe complex, essentially identical to that obtained for the EH and E forms of the native enzyme. Addition of azide to ZnCPD.L-Phe at pH 7 markedly changes the zinc coordination sphere from 4 N/O atoms at 2.021 +/- O.06 A and 1.4 +/- 0.5 N/O atoms at 2.54 +/- 0.5 A to 3.9 N/O atoms at 1.995 +/- 0.006 A. The decrease in R of about 0.03 A in both the Zn- and CoCPD.L-Phe.N3-complexes is likely due to the ligand exchange from a neutral water to an anion. The XAFS spectra of the ternary complex is pH independent from 7 to 9, in agreement with the ionization of the water being the source of the spectral changes in the free enzyme and its binary L-Phe complex. The enzyme.azide.L-Phe complex is likely bound in a manner analogous to that expected for a post-transition state in a biproduct complex for peptide hydrolysis--that is, the carboxylate anion of the peptide bound to the Zn and the protonated form of L-Phe H-bonded to the catalytic Glu-270 carboxylate. The XAFS results on the "spectroscopically silent" ZnCPD are compared to nuclear magnetic resonance and electron absorption studies on CoCPD.
Zhang
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K
Biostructure Institute, University City Science Center, University of Pennsylvania, Philadelphia 19104, USA.
Auld
D S
DS
D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry*; D043422:Carboxypeptidases A; D002417:Cattle; D003035:Cobalt / Q000737:chemistry*; D007477:Ions; D008956:Models, Chemical; D013057:Spectrum Analysis; D014867:Water / Q000737:chemistry; D014965:X-Rays; D015032:Zinc / Q000737:chemistry*
D007477:Ions; D014867:Water; D003035:Cobalt; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc