STRUCTURAL INVESTIGATIONS ON THE COORDINATION ENVIRONMENT OF THE ACTIVE-SITE COPPER CENTERS OF RECOMBINANT BIFUNCTIONAL PEPTIDYLGLYCINE ALPHA-AMIDATING ENZYME

摘要

The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative, The enzyme contains 2 coppers per 75 kDa protein molecule, Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 +/- 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu:(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the non-histidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/Cl ligand per copper, The value of v(CO) (2093 cm(-1)) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the Delta Pro-PHM382s mutant M(314)I is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val-Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound-reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to CUAI and 2 histidines and 1 methionine as ligands to CUBI. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CUA is also entirely consistent with the data.