289 - Kinetics and biological consequences of quinone-induced protein adduction

Abstract

Background Quinone groups are a common motif in many biological compounds and have been linked which tissue damage as a result of their capacity to undergo redox cycling to generate radicals, and/or by acting as Michael acceptors with nucleophiles, such as Cys residues on proteins, with consequent adduct formation and aggregation. The kinetics and consequences of these reactions are poorly characterized. Hypothesis That Michael addition reactions of quinones with Cys residues on proteins are rapid, structure dependent, and quantitatively significant. Furthermore, these reactions induce altered protein structure and function. Results A range of quinones were incubated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), creatine kinase (CK), papain, bovine serum albumin (BSA), and human serum albumin (HSA), with the kinetics of adduction and effects on protein structure and activity determined. The rate constants, k, for adduction at Cys residues, were dependent on the thiol pKa, and quinone and protein structure, and are 102-105 M-1s-1. Some quinones induced protein dimerization (GAPDH and CK, but not BSA, HSA, or papain) in a dose- and time-dependent manner. Incubation of quinones with purified GAPDH and CK, or cell lysates, resulted in rapid and extensive loss of enzyme activity. The rate constants for quinone adduction correlate strongly with loss of activity. GSH reacts rapidly, and competitively, with some quinones, and can prevent/reverse loss of activity and protein dimerization. Mass spectrometry with peptide mass mapping shows that benzoquinone selectively forms adducts at Cys149 and Cys244 with GAPDH, but modifies all Cys residues in CK. Conclusions These data indicate that quinones can rapidly and selectively modify some proteins via adduct formation at Cys residues, with this altering protein structure and function. The rate constants for these quinone-induced modifications at Cys residues are higher than those observed with a number of other important biological oxidants including H2O2 and O2•–.