title: The effect of deleting a putative salt bridge on the properties of the thermostable subtilisin-like proteinase, aqualysin I.
abstract: Aqualysin I, is a subtilisin-like serine proteinase, from the thermophilic bacterium Thermus aquaticus. It is predicted that the enzyme contains a salt bridge, D17-R259, connecting the N- and C-terminal regions of the enzyme. Previously we reported on the stabilizing effect of the incorporation of a salt bridge at a corresponding site in VPR, a related cold adapted enzyme from a marine Vibrio sp. Here we describe the effect of the reverse change, i.e. the elimination of the salt bridge on the thermal stability and kinetic properties of aqualysin I. Deletion of the putative salt bridge in the D17N mutant of the enzyme destabilized the enzyme by 8-9 °C in terms of T₅₀%, determined by thermal inactivation and over 4 °C in T(m), as measured from melting curves of the inhibited enzyme. The mutation, however, had no significant effect on the kinetic parameters of the enzyme under standard assay conditions.
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavík, Iceland.
authors.forename: Jóhanna
authors.initials: J
authors.lastname: Arnórsdóttir
authors.forename: Manuela
authors.initials: M
authors.lastname: Magnúsdóttir
authors.forename: Olafur H
authors.initials: OH
authors.lastname: Friđjónsson
authors.forename: Magnús M
authors.initials: MM
authors.lastname: Kristjánsson
mesh_terms: D000222:Adaptation, Physiological; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013824:Thermus / Q000201:enzymology
chemical_list: D012697:Serine Endopeptidases; C055102:aqualysin I