A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 2922 - 2933
• Main Authors: Costa, Evenilton P., Façanha, Arnoldo R., Cruz, Criscila S., Silva, Jhenifer N., Machado, Josias A., Carvalho, Gabriel M., Fernandes, Mariana R., Martins, Renato, Campos, Eldo, Romeiro, Nelilma C., Githaka, Naftaly W., Konnai, Satoru, Ohashi, Kazuhiko, Vaz, Itabajara S., Logullo, Carlos
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: active sites; Amino Acids - metabolism; Animals; ascorbic acid; Calcium - pharmacology; Cooperativity; cysteine; Disulfide bond formation; disulfide bonds; Disulfides - metabolism; Electrophoresis, Polyacrylamide Gel; Embryogenesis; Fluorides - pharmacology; glutathione; Glutathione Disulfide - metabolism; hydrogen peroxide; Hydrophobic and Hydrophilic Interactions; hydrophobic bonding; inorganic pyrophosphatase; Inorganic Pyrophosphatase - metabolism; Kinetics; Metabolism; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins - metabolism; orthophosphates; Oxidants - pharmacology; Oxidation-Reduction; physicochemical properties; Protein Multimerization - drug effects; Pyrophosphatase; recombinant proteins; Recombinant Proteins - metabolism; Reducing Agents - pharmacology; Sulfhydryl Compounds - metabolism; thermodynamics; Thiol redox status; thiols; ticks; Ticks - enzymology; Disulfide bond formation; Thiol redox status; Embryogenesis; Cooperativity; Metabolism; Pyrophosphatase
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.09.017

Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status. [Display omitted] •A pyrophosphatase is regulated by dimeric cooperativity according to the cysteine redox status.•The results are carefully addressing the participation of pyrophosphatase during embryogenesis.•A new angle of redox control by disulfide bond formation on enzymatic regulation