STRUCTURE AND DYNAMICS OF THE METAL SITE OF CADMIUM-SUBSTITUTED CARBOXYPEPTIDASE-A IN SOLUTION AND CRYSTALLINE STATES AND UNDER STEADY-STATE PEPTIDE HYDROLYSIS

PAC spectra (perturbed angular correlation of gamma-rays) of cadmium-s ubstituted carboxypeptidase A (CPD) show that the enzyme in solution i mposes a flexible, pH-and chloride-dependent coordination structure on the metal site, in contrast to what is found in the crystalline state . A much more restricted coordination geometry occurs for the steady-s tate peptide intermediates of Bz-Gly-L-Phe and Bz-Gly-L-Phe in solutio n, suggesting that substrate binding locks the structure in a rigid co nformation. The results further indicate that the peptide intermediate has a six-coordinated metal coordination geometry with an OH- ligand at the solvent site and a carbonyl oxygen at an additional ligand site . In marked contrast, conformational rigidity is not induced by the in hibitor/poor substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly, Bz-Gly-Gly, and L-Phe. These results are consisten t with an intact scissile peptide bond in the enzyme-substrate complex of Bz-Gly-L-Phe and Bz-Gly-Gly-L-Phe. A single nuclear quadrupole int eraction (NQI) is observed for the crystalline state of the enzyme bet ween pH 5.7 and pH 9.4. This NQI agrees with calculations based on the metal coordination geometry for cadmium in crystalline CPD derived fr om X-ray diffraction studies. A single broad distribution of NQIs is o bserved for CPD in sucrose solutions and 0.1 M NaCl at pH values below 6.5. This NQI (NQI-1') has parameters very close to those for the cry stalline state. The enzyme metal site, characterized by this NQI, is c onverted into two new enzyme metal sites over the pH range of 6.5-8.3. The metal coordination sphere of one of these has a NQI (NQI-1) with parameters similar to those at lower pH values (NQI-1') while the othe r NQI (NQI-2) is characterized by markedly different NQI parameters. A ngular overlap model (AOM) calculations indicate that the coordination sites giving NQI-1' and NQI-1 both have a metal-bound water molecule while the coordination site giving NQI-2 has a metal-bound hydroxide i on, PAC results at pH 8.3-10.5 indicate that in this pH range the two metal coordination geometries related to NQI-1 and NQI-2 occur in a pH independent ratio of 2:1, with the one with the water ligand being th e most abundant species. The observed pH-independent equilibrium betwe en the two different metal coordination geometries for cadmium can be explained by an equilibrium between tautomeric forms of a hydrogen bon d between the Glu-270 carboxyl group and the metal-bound water (Glu-27 0 COO-...(HOH)M reversible arrow Glu-270 COOH ...(OH-)M) being slow on the time scale of a PAC experiment, i.e., slower than 0.5 mu s. We fi nally suggest that NQI-1' observed at low pH reflects an enzyme specie s containing a metal-coordinated water molecule and the protonated car boxyl group of Glu-270.