Activation of Helicobacter pylori inorganic pyrophosphatase and the importance of Cys16 in thermostability, enzyme activation and quaternary structure.
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.
Lee
Mon-Juan
MJ
Institute of Biotechnology, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu, 30013, Taiwan.
Huang
Haimei
H
Lin
Wei
W
Yang
Ray-Rong
RR
Liu
Chien-Liang
CL
Huang
Chung-Yu
CY
D003545:Cysteine; D004789:Enzyme Activation; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary
D043564:Inorganic Pyrophosphatase; D003545:Cysteine