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 organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, 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 reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.
Paoli
P
P
Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze 50134, Italy.
Giannoni
E
E
Pescitelli
R
R
Camici
G
G
Manao
G
G
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D011061:Poly A / Q000378:metabolism; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D004220:Disulfides; D006861:Hydrogen Peroxide; D011061:Poly A; D000097024:Acylphosphatase