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.
Arnórsdóttir
Jóhanna
J
Deparment of Biochemistry, Science Institute, University of Iceland, 107 Reykjavík, Iceland.
Sigtryggsdóttir
Asta Rós
AR
Thorbjarnardóttir
Sigrídur H
SH
Kristjánsson
Magnús M
MM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003080:Cold Temperature; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000235:genetics*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
D050505:Mutant Proteins; D011994:Recombinant Proteins; D011392:Proline; D012697:Serine Endopeptidases; D013381:Subtilisins; C055102:aqualysin I