An XAS investigation of product and inhibitor complexes of Ni-containing GlxI from Escherichia coli: mechanistic implications.
Escherichia coli glyoxalase I (GlxI) is a metalloisomerase that is maximally activated by Ni(2+), unlike other known GlxI enzymes which are active with Zn(2+). The metal is coordinated by two aqua ligands, two histidines (5 and 74), and two glutamates (56 and 122). The mechanism of E. coli Ni-GlxI was investigated by analyzing Ni K-edge X-ray absorption spectroscopic (XAS) data obtained from the enzyme and complexes formed with the product, S-D-lactoylglutathione, and various inhibitors. The analysis of X-ray absorption near edge structure (XANES) was used to determine the coordination number and geometry of the Ni site in the various Ni-GlxI complexes. Metric details of the Ni site structure were obtained from the analysis of extended X-ray absorption fine structure (EXAFS). Interaction of S-D-lactoylglutathione (product) or octylglutathione with the enzyme did not change the structure of the Ni site. However, analysis of XAS data obtained from a complex formed with a peptide hydroxamate bound to Ni-GlxI is consistent with this inhibitor binding to the Ni center by displacement of both water molecules. XANES analysis of this complex is best fit with a five-coordinate metal and, given the fact that both histidine ligands are retained, suggests the loss of a glutamate ligand. The loss of a glutamate ligand would preserve the neutral charge on the Ni complex and is consistent with the lack of a significant shift in the Ni K-edge energy in this complex. These data are compared with data obtained from the E. coli Ni-GlxI selenomethionine-substituted enzyme. The replacement of three methionine residues in the native enzyme with selenomethionine does not affect the structure of the Ni site. However, addition of the peptide hydroxamate inhibitor leads to the formation of a complex whose structure as determined by XAS analysis is consistent with inhibitor binding via displacement of both water molecules but retention of both histidine and glutamate ligands. This leads to an anionic complex, which is consistent with an observed 1.7 eV decrease in the Ni K-edge energy. Plausible reaction mechanisms for Ni-GlxI are discussed in light of the structural information available.
Davidson
G
G
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Clugston
S L
SL
Honek
J F
JF
Maroney
M J
MJ
D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry; D006877:Hydroxamic Acids / Q000737:chemistry; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D009532:Nickel / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D012542:Scattering, Radiation; D013057:Spectrum Analysis / Q000379:methods; D014965:X-Rays
D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D008024:Ligands; D046911:Macromolecular Substances; D009842:Oligopeptides; C059196:S-octylglutathione; D009532:Nickel; D007791:Lactoylglutathione Lyase; D005978:Glutathione; C013585:S-lactoylglutathione