Site Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Proteome Damage Inflicted by Ionizing Radiation: Advancing a Theme in the Research of Miroslav Radman

Oxidative proteome damage has been implicated as a major contributor to cell death and aging. Protein damage and aging has been a particular theme of the recent research of Miroslav Radman. However, the study of how cellular proteins are damaged by oxidative processes is still in its infancy. Here we examine oxidative changes in the proteomes of four bacterial populations—wild type E. coli, two isolates from E. coli populations evolved for high levels of ionizing radiation (IR) resistance, and D. radiodurans—immediately following exposure to 3000 Gy of ionizing radiation. By a substantial margin, the most prominent intracellular oxidation events involve hydroxylation of methionine residues. Significant but much less frequent are carbonylation events on tyrosine and dioxidation events on tryptophan. A few proteins are exquisitely sensitive to targeted oxidation events, notably the active site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in E. coli. Extensive experimental evolution of E. coli for IR resistance has decreased overall proteome sensitivity to oxidation but not to the level seen in D. radiodurans. Many observed oxidation events may reflect aspects of protein structure and/or exposure of protein surfaces to water. Proteins such as GAPDH and possibly Ef-Tu may have an evolved sensitivity to oxidation by H2O2.

Modelling the active site of glyceraldehyde-3 phosphate dehydrogenase with the LSCF formalism

In the framework of a theoretical approach to the relationship between structure and reactivity of the catalytic centers of enzymes, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) has been chosen as a model enzyme. In GAPDH, the proximity of His176 increases the reactivity of Cys149 at neutral pH; however, its presence alone is not sufficient to explain the reactivity of the catalytic Cys. In order to determine which other interactions play an important role, a study of the geometric and electronic structure of the catalytic site has been made using a hybrid quantum mechanics/molecular mechanics local self-consistent field method. This allows the computation of the electronic properties of amino acid residues in subsystems influenced by other parts of the macromolecule. The quantum subsystem was centered on the Cys149 residue of GAPDH. The structures of GAPDH taken from the crystallographic database did not include hydrogen atoms and these had to be added taking into account the fact that, in the active site, His176 has three tautomeric forms: δ-His protonated, e-His protonated and His+. The results presented here suggest that the most stable His…Cys system in GAPDH is a strongly hydrogen-bonded Cys149 −/His176 + ion pair.

STUDIES ON THE BIOCHEMICAL BASIS OF DISTAL AXONOPATHIES–I. INHIBITION OF GLYCOLYSIS BY NEUROTOXIC HEXACARBON COMPOUNDS

Abstract— Neurotoxic hexacarbon compounds 2,5‐hexanedione (2,5‐HD) and methyl n‐butyl ketone (MnBK) inhibit crystalline and endogenous CNS and PNS glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Preincubation of the enzyme with the toxin was necessary for inhibition. The enzyme activity of GAPDH was preserved by the addition of dithiothreitol in the presence of either neurotoxin. By contrast, lactate dehydrogenase (LDH) activity was not inhibited by these neurotoxic chemicals. Neurologically inactive compounds 1,6‐hexanediol and acetone failed to inhibit GAPDH. The present data indicate that 2,5‐HD and M “BK block energy metabolism by inhibiting glycolysis at the site of GAPDH. These observations may account for the known failure of GAPDH‐dependent axonal transport and the axonal degeneration which occurs in hexacarbon neuropathy.

Introduction of an intramolecular crosslink at the active site of glyceraldehyde 3-phosphate dehydrogenase

Reaction of rabbit muscle apo-glyceraldehyde 3 -phosphate dehydrogenase with one mole of 1, 5-difluoro, 2, 4-dinitrobenzene per mole of enzyme protomer brings about total loss of enzymatic activity and concomitant introduction of covalent intramolecular crosslinks at the active site of the enzyme. Following peptic digestion, a crosslinked couple of peptides was purified and found to have the structure: ▪ The crosslinked cysteine and lysine residues, though some 32 amino acid residues apart in the primary sequence, may approach each other to a distance of 5–6 Å in the three dimensional structure of the enzyme.