Activation of Helicobacter pylori inorganic pyrophosphatase and the importance of Cys16 in thermostability, enzyme activation and quaternary structure

Abstract

The inorganic pyrophosphatase from the human pathogen Helicobacter pylori (HpPPase) is a family I PPase. It is a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of inorganic pyrophosphate (PP(i)) by HpPPase relied on the presence of magnesium and followed Michaelis-Menten kinetics, with k (cat) being 344 s(-1) and K (m) being 83 microM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substitution of Cys16 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C16S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y140F HpPPase and those of Escherichia coli. Although Cys16 was not located at the subunit interface of the hexameric HpPPase, sedimentation analysis results suggested that the C16S substitution destabilized HpPPase through impairing trimer-trimer interactions. This study provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure.

The inorganic pyrophosphatases of microorganisms: a structural and functional review

“…This enzyme may represent a novel target for treatment. When nonconserved Cys16 is substituted with serine, it loses 50% of its activity, has reduced sensitivity to reducing agents, and experiences a decline in thermostability ( Lee et al, 2007 ). The effect on the active site is related to the environment surrounding Cys 16 in the active site, with the valine residues forming a hydrophobic cluster.…”

Metalloregulation of Helicobacter pylori physiology and pathogenesis

“…Pyrophosphatases are ubiquitious enzymes that catalyze the interconversion of inorganic pyrophosphate and orthophosphate, and function as a catalyst within cellular bioenergetics, making this group of enzymes essential for life. The pyrophosphatases within H. pylori requires Mg 2+ for enzymatic activity (Lee et al, 2007 ). In addition to its role as a cofactor within H. pylori phosphate metabolism, Mg 2+ may also play an important role in H. pylori transcriptional regulation through the binding of a lower affinity site within the transcription factor NikR.…”