NMR and computer modeling studies of the conformations of glutathione derivatives at the active site of glyoxalase I

• Published in: The Journal of biological chemistry Vol. 259; no. 18; pp. 11436 - 11447
• Main Authors: Rosevear, P R, Sellin, S, Mannervik, B, Kuntz, I D, Mildvan, A S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.09.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Computers; Enzymes and enzyme inhibitors; Erythrocytes - enzymology; Fundamental and applied biological sciences. Psychology; Glutathione - analogs & derivatives; Glutathione - metabolism; Humans; Lactoylglutathione Lyase - metabolism; Lyases; Lyases - metabolism; Magnetic Resonance Spectroscopy; Manganese - metabolism; Mathematics; Models, Molecular; Protein Conformation; Zinc - metabolism; Human; Enzyme; Active site; Substrate specificity; Red blood cell; Molecular interaction; Lactoyl-glutathione lyase; NMR spectrometry; Metal enzyme complex; Conformation; Glutathione
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)90880-X

The conformations of four derivatives of glutathione bound at the active site of the metalloenzyme glyoxalase I have been determined by NMR measurements and by computer model building using a distance geometry approach. Paramagnetic effects of Mn2+-glyoxalase I on the longitudinal relaxation rates of the carbon-bound protons of the substrate analog S-(acetonyl)-glutathione at three frequencies, the hydrophobic competitive inhibitor S-(propyl)glutathione at four frequencies, and the charged competitive inhibitor S-(carboxymethyl)glutathione at a single frequency were used to calculate Mn2+ to proton distances in each complex. These and previously determined distances from Mn2+ to the protons and 13C-enriched carbon atoms of the product S-(D-lactoyl)glutathione were used in a distance geometry program to compute the conformations of each enzyme-bound derivative which best fit the measured distances and other known constraints such as bond lengths, van der Waals radii, planar and trans-peptide bonds, and thioester linkages. The distance geometry program also provided a measure of the uniqueness of the conformations consistent with the experimental data. Extended Y-shaped conformations were detected for each of the bound glutathione derivatives, similar to the x-ray structure and the theoretically calculated conformation of glutathione itself, suggesting this to be a low energy form. Acceptable conformations of each enzyme-bound derivative fell into two classes with the metal either above or below the mean plane through the glutathione compound. The conformational uncertainty within each class was relatively small, ranging from deviations of 0.9-1.9 A in the average positions of each of the atoms. A small but significant difference in the conformation of the substrate analog as compared to the product was detected in the position of the reaction center carbon directly bonded to the glutathione sulfur atom. Unlike the second-sphere metal complexes formed by the bound substrate analog, the product, or the hydrophobic competitive inhibitor, the charged competitive inhibitor S-(carboxymethyl)glutathione binds farther from the metal, in the third coordination sphere.