Effect of proline substitutions on stability and kinetic properties of a cold adapted subtilase.

A cold adapted subtilisin-like serine proteinase from a Vibrio species is two amino acids shorter at the N-terminus than related enzymes adapted to higher temperatures and has a 15 residues' C-terminal extension relative to the highly homologous thermophilic enzyme aqualysin I from Thermus aquaticus. These enzymes are produced as pro-enzymes with an N-terminal chaperone sequence for correct folding and a C-terminal signal peptide for secretion, which are subsequently cleaved off by autocatalysis to give the mature enzyme. A truncated form of the Vibrio proteinase where the C-terminal extension was removed and two residues near the N-terminus were substituted with proline, to resemble the N- and C-terminal regions in aqualysin I, resulted in increased thermostability and diminished catalytic efficiency. The proline substitutions shift the site of autocatalytic cleavage at the N-terminus by two amino acids, apparently by rigidifying the terminal residues and support the formation of a beta-sheet that fixes the N-terminus to the main body of the protein.