Electron paramagnetic resonance study of the active site of copper-substituted human glyoxalase I.

TLDR: It is demonstrated that at least one nitrogen ligand is involved in the binding of Cu2+ and a good fit was obtained for the low-field region of the asymmetric spectrum of free 63Cu(II) glyoxalase I.

Abstract: Zn/sup 2 +/ in native glyoxalase I from human erythrocytes can be replaced by Cu/sup 2 +/, giving an inactive enzyme. Cu/sup 2 +/ was demonstrated to compete with the activating metals Zn/sup 2 +/ and Mn/sup 2 +/, indicating a common binding site on the enzyme for these metal ions. The electron paramagnetic resonance (EPR) spectra of /sup 63/Cu(II) glyoxalase I at 77 K and of its complexes with glutathione and some glutathione derivatives are characteristic of Cu/sup 2 +/ in an elongated octahedral coordination. The low-field bands of the free enzyme are asymmetric and become symmetrical upon addition of glutathione of S-(p-bromobenzyl) glutathione but not S-(D-lactoyl)glutathione. The results indicate the existence of two conformations of Cu(II) glyoxalase I, in agreement with the effects caused by these compounds on the protein fluorescence. The copper hyperfine line at low field in the EPR spectrum of the S-(p-bromobenzyl) glutathione complex of /sup 63/Cu(II) glyoxalase I shows a triplet structure, indicative of coupling to one nitrogen ligand in the equatorial plane. By addition of the spectrum of the S-(p-bromobenzyl)glutathione complex and a spectrum corresponding to two nitrogen ligands with two different coupling constants, a good fit was obtained for the low-field regionmore » of the asymmetric spectrum of free /sup 63/Cu(II) glyoxalse I. The results demonstrate that at least one nitrogen ligand is involved in the binding of Cu/sup 2 +/. A hypothetical model for the metal coordination site of glyoxalase I with different conformations of the enzyme is presented. Model building, using distances from earlier proton NMR studies of glutathione derivatives, showed that it is sterically possible for an imidazole group of histidine in an apical coordination position to act as the base in the catalytic mechanism.« less