Nonredox nickel enzymes.

ion as well as other steps contributed to the mechanism Figure 11. Sequence alignments of selected class I and class II glyoxalase I enzymes created using Clustal W2. Amino acids are colored by property (hydrophobic (red), acidic (blue), basic (purple), other (green)). Metal binding residues are highlighted in yellow. Residues marked with an asterisk (∗) are invariant; thosemarked by other symbols represent low (:) andmoderate (.) variability. TheN-terminal extension and additional loops found in class I enzymes are highlighted in blue. The S. cerevisiae sequence was truncated after 226 of 326 residues. Chemical Reviews Review dx.doi. org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4218 of product formation. One possible explanation for the difference between the Ni(II)and Cd(II)-bound enzyme kinetics is that differences in the extent of polarization of the substrate result from different metals in the active site, with Cd(II) being less polarizing and therefore less efficient at lowering the energy barrier for proton abstraction. Differences in aqua ligand exchange rates may also play a role, particularly for the Ni(II) enzyme where a slower on-rate for the substrate might suppress the observation of the KIE. Once formed, the enediol(ate) can be protonated at the alternate C atom to complete the isomerization. Based in part on crystal structures of inhibitor complexes (vide supra), mechanisms involving coordination of the enediolate have been proposed (Figure 14), as have mechanisms that involve activatingmetal-bound water molecules to serve as catalytic bases without binding the substrate to the metal. The crystal structures reveal the apparent importance of two cis-aqua ligands in the active site, which could indicate the need for two ciscoordination positions, or a mechanism that employs proton transfers involving both aqua ligands. Coordination of the substrate with loss of a carboxylate ligand has the attractive feature that the Ni(II) site is always charge neutral, and consistent with model chemistry. Efforts to distinguish these mechanisms have employed kinetics and XAS studies of the E. coli Ni(II) enzyme in solution in the presence of various inhibitors, mutations, etc., and the crystal structure of human Glo I obtained in the presence of the transition state analogue, S(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione. The latter structure reveals a five-coordinate Zn center in which both water molecules and Glu172 are displaced upon binding the inhibitor, which binds in a bidentate fashion. Glu172 was proposed to play a role as the catalytic base in the deprotonation and reprotonation of the substrate, a role that is consistent with computational models. Both Rand S-enantiomers of substrates are bound and stereospecifically reprotonated. The presence of two chemically equivalent Glu ligands suggests that one (Glu172) might be the catalytic base for the S-enantiomer, while the other (Glu99) serves that role for the Renantiomer. The corresponding residues in the E. coli enzyme are Glu122 and Glu56, and XAS studies of a hydroxamate inhibitor (L-γ-glutamyl-N-hydroxy-N-methyl-L-glutaminylglycine) complex are consistent with substitution of both aqua ligands, although evidence for the release of a carboxylate was not specifically observed. Although changes in metal ligands greatly affect metal binding affinities, mutation of Glu56 in E56A-E. coli Glo I resulted in the reduction of enzymatic activity to <4% of wild type under conditions of metal saturation, consistent with similar mutations of class I enzymes and the putative role for the carboxylate as a catalytic base. Figure 12. Ribbon diagram of the crystal structure of E. coliGlo I, (PDB code 1F9Z) showing the two subunits of the homo dimer in cyan and gray and the location of the two Ni sites (green spheres) at subunit interfaces. Figure 13.Comparison of the metal site structure of the Ni(II) complex (panel A, PDB code 1F9Z) and the Zn(II) complex (panel B, 1FA5) of E. coli Glo I showing the change in coordination number and geometry for the two metals. The nickel and zinc ions are represented in gray and dark blue, respectively, while CPK coloring is used for all other atoms. WAT = solvent molecules. Protein residues are distinguished by letters indicating the two different subunits of the enzyme. Chemical Reviews Review dx.doi. org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4219