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α,β-Unsaturated carbonyls make up 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 data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 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 (K_I) (i.e., ACR ≪ MVK < acrolein) was related to the calculated …