Disulfide-mediated dimerization stabilizes pyruvate kinase of Cryptosporidium parvum: Insights into unique structural...

Cryptosporidium parvum is a protozoan parasite that depends mainly on glycolysis for energy production. C. parvum pyruvate kinase (CpPyK), which catalyzes the final step of glycolysis, contains covalently cross-linked dimers generated by the formation of an intermolecular disulfide (SS) bond between Cys26 and Cys312 of two CpPyK monomers. This bridge is unique in CpPyK and not present in other known pyruvate kinases. To investigate the effect of this SS-mediated dimerization on the structure and function of the enzyme, we generated a Cys312Ser mutant and compared the structural and kinetic properties of the resulting mutant CpPyK (mCpPyK) to wild-type CpPyK (wtCpPyK). While wtCpPyK assembles as a tetramer, mCpPyK exists in multiple states including the tetrameric species. Differential scanning fluorimetry (DSF) revealed that wtCpPyK unfolds with a single melting transition at ∼67 °C. However, mCpPyK exhibits multiple peaks along the unfolding pathway with transitions at 36 °C, 48 °C, and 60 °C. Addition of effectors such as phosphoenolpyruvate (PEP), glucose-6-phosphate (G6P), and adenosine monophosphate (AMP) to mCpPyK consolidated these transitions and increased the melting temperature of mCpPyK to as high as ∼65 °C. Enzyme kinetic analyses of mCpPyK showed reduced catalytic turnover for PEP by approximately 30% (kcat ∼378 min^-1 in wtCpPyK versus ∼269 min^-1 in mCpPyK) and different allosteric responsiveness. These results demonstrate that the SS cross-linkage in CpPyK contributes to structural integrity and modulates the enzyme's allosteric responsiveness. We propose that this covalent adaptation aids protection of the enzymatic function under environmental stress providing an evolutionary advantage for the parasite.