title: Effects of metal ions on stability and activity of hyperthermophilic pyrolysin and further stabilization of this enzyme by modification of a Ca2+-binding site.
abstract: Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na(+), Ca(2+), or Mg(2+) salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca(2+)-binding sites (Ca1 and Ca2) revealed that the binding of Ca(2+) is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca(2+)-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Gao
authors.forename: Zheng
authors.initials: Z
authors.lastname: Dai
authors.forename: Bing
authors.initials: B
authors.lastname: Tang
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D008670:Metals; D012697:Serine Endopeptidases; C101133:pls protein, Pyrococcus furiosus; D002118:Calcium