Molecular Mechanism of Glyceraldehyde-3-phosphate Dehydrogenase Inactivation by α,β-Unsaturated Carbonyl Derivatives

Abstract

α,β-Unsaturated carbonyls are an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these datagaps, purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was incubated with acrylamide (ACR), acrolein or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration-and time-dependent manner. The rank order of enzyme inhibition (KI); i.e., ACR << MVK < acrolein, was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pKa-lowering microenvironment (pKa = 6.03) and, at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate-state. These data suggest a general cytotoxic mechanism where electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, since chemical derivitization causes loss of protein function.

Acrolein, an α,β-Unsaturated Aldehyde, Irreversibly Inhibits the Acetylation of Aromatic Amine Xenobiotics by Human Arylamine N-Acetyltransferase 1

“…Similar results have been recently reported for other families of enzymes such as human protein tyrosine phosphatase 1B, protein disulfide isomerase, and glyceraldehyde 3-phosphate dehydrogenase, which are irreversibly inhibited by ACR through covalent Michael adducts with their catalytic cysteine residue (Carbone et al, 2005;Seiner et al, 2007;Martyniuk et al, 2011;Nakamura et al, 2013). In aqueous solution, thiols react with ACR through conjugate addition to the double bond of the a,b-unsaturated aldehyde (thus forming a Michael adduct) rather than by addition to the aldehyde moiety (Seiner et al, 2007).…”

“…Its cellular toxicity relies mainly on its ability to deplete glutathione and to form DNA and protein adducts (Cai et al, 2009). Formation of ACR-protein covalent adducts is principally due to a Michael addition where the b-carbon of acrolein reacts with nucleophilic groups to form the 1,4 addition with the double bond (Cai et al, 2009;Martyniuk et al, 2011).…”

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

“…Oxidation of Cys 358 of PKM2 was previously found to result in inhibition of enzymatic activity (65); therefore, our finding of ARF-induced inhibition of PK activity and oxidation of Cys 358 of PK are consistent with this previous study. Additionally, our finding of increased oxidation in Cys 247 of glyceraldehyde 3-phosphate dehydrogenase (G3P2, 2.3-fold) mediated by ARF is also interesting because modification of this residue is also correlated with decreased enzyme activity (66). Collectively, these findings indicate that the Trx redox system plays an important role in glycolysis by regulating redox states of Cys residues of proteins involved in the glycolytic enzymatic pathways.…”

Target for Diverse Chemical Modifications

“…Two of these three lipid peroxidative by-products, namely acrolein (O¼CH-CH¼CH 2 ) and 4-hydroxy-2-nonenal (O¼CH-CH¼CH-CHOH-(CH 2 ) 4 -CH 3 ), have been shown to react with GAPDH [32,33,[74][75][76]. Additionally, GAPDH was detected as a cellular target of hydroxynonenal in a study [77] that used renal tubular epithelial cells.…”

“…Martyniuk and coworkers [75] examined the effects of acrolein and two other environmental toxins (i.e. methylvinylketone and acrylamide) on the activity of GAPDH.…”