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Journal of Biochemistry Advance Access originally published online on December 12, 2008
Journal of Biochemistry 2009 145(3):325-329; 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 Arnórsdóttir1, Ásta Rós Sigtryggsdóttir1, Sigríður H. Thorbjarnardóttir2 and Magnús M. Kristjánsson1,{dagger}

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

{dagger}To whom correspondence should be addressed. 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

Abbreviations: PMSF, phenylmethanesulfonyl fluoride; Suc-AAPF-NH-Np, Succinyl-AlaAlaProPhe-p-nitroanilide; VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species

*Enzyme: Vibrio-proteinase (EC 3.4.21.-)


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