Reaction of quinones with proteins: Kinetics of adduct formation, effects on enzymatic activity and protein structure, and potential reversibility of modifications

Quinones are a common motif in many biological compounds, and have been linked to tissue damage as they can undergo redox cycling to generate radicals, and/or act as Michael acceptors with nucleophiles, such as protein Cys residues, with consequent adduct formation. The kinetics and consequences of these Michael reactions are poorly characterized. In this study we hypothesized that adduction of protein Cys residues with quinones would be rapid, structure-dependent, quantitatively-significant, and result in altered protein structure and function. Multiple quinones were incubated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), creatine kinase (CK), papain, bovine (BSA) and human (HSA) serum albumins, with the kinetics of adduction and effects on protein structure and activity determined. Adduction rate constants at Cys residues, which were dependent on the quinone and protein structure, and thiol pKa, are in the range 102-105 M−1 s−1. p-Benzoquinone (BQ) induced dimerization of GAPDH and CK (but not BSA, HSA, or papain) in a dose- and time-dependent manner. Incubation of purified proteins, or cell lysates, with quinones resulted in a rapid loss of GAPDH and CK activity; this loss correlated well with the rate constant for Cys adduction. Glutathione (GSH) reacts competitively with quinones, and could reverse the loss of activity and dimerization of GAPDH and CK. Mass spectrometry peptide mass mapping provided evidence for BQ adduction to GAPDH to specific Cys residues (Cys149, Cys244), whereas all Cys residues in CK were modified. These data suggested that quinones can induce biological effects by rapid and selective formation of adducts with Cys residues in proteins. [Display omitted] •Quinone toxicity has been linked to redox cycling or Michael adduction reactions.•Rate constants for Michael adduction to Cys in proteins are rapid (102-105 M−1s−1).•Reaction at Cys inactivates enzymes and generates quinone-linked dimers.•GSH reacts rapidly with quinones and can inhibit or reverse damage to some enzymes.•Cys modification by quinones is more rapid than that induced by many common oxidants.