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Journal of Biochemistry Advance Access published online on December 12, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn168
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© The authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

Jóhanna Arnórsdóttir, Ásta Rós Sigtryggsdóttir, Sigríður H. Thorbjarnardóttir2 and Magnús M. Kristjánsson1

1Deparment of Biochemistry, Science Institute, University of Iceland, 107 Reykjavík, Iceland.
2Institute of Biology, University of Iceland, 101 Reykjavík, Iceland.

Correspondence to Magnús M. Kristjánsson, Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavík, Iceland. Tel.: +354 525 4800. Fax: +354 552 8911. E-mail: mmk{at}raunvis.hi.is

Received September 8, 2008; Accepted December 1, 2008


  Abstract

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 β-sheet that fixes the N-terminus to the main body of the protein.

Key Words: kinetic properties, psychrophilic, proline, site directed mutagenesis, stability


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