Hydrogen peroxide triggers the formation of a disulfide dimer of muscle acylphosphatase and modifies some functional properties of the enzyme

Acylphosphatase is expressed in vertebrates as two molecular forms, the org an common and the muscle types. The former does not contain cysteine residu es, whereas the latter contains a single conserved cysteine (Cys-21). We de monstrated that H2O2 at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties d iffering from those of the reduced enzyme. In particular, we observed chang es in the kinetic behavior of its intrinsic ATPase activity, whereas the ki netic behavior of its benzoyl phosphatase activity does not change. Moreove r, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the red uced enzyme does not bind polynucleotides. Experiments performed with H2O2 in the presence of increasing SDS concentrations demonstrated that disulfid e dimer formation is prevented by SDS concentrations higher than 300 mum, s uggesting that a non-covalently-linked dimer is present in non-denaturing s olvents. Light-induced cross-linking experiments performed on the Cys-21 Se r mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-li nked dimer is in fact present in non-denaturing solutions and that an enzym e group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.