Effect of selected Ser/Ala and Xaa/Pro mutations on the stability and catalytic properties of a cold adapted subtilisin-like serine proteinase.
A subtilisin-like serine proteinase from a psychrotrophic Vibrio species (VPR) shows distinct cold adapted traits regarding stability and catalytic properties, while sharing high sequence homology with enzymes adapted to higher temperatures. Based on comparisons of sequences and examination of 3D structural models of VPR and related enzymes of higher temperature origin, five sites were chosen to be subject to site directed mutagenesis. Three serine residues were substituted with alanine and two residues in loops were substituted with proline. The single mutations were combined to make double and triple mutants. The single Ser/Ala mutations had a moderately stabilizing effect and concomitantly decreased catalytic efficiency. Introducing a second Ser/Ala mutation did not have additive effect on stability; on the contrary a double Ser/Ala mutant had reduced stability with regard to both wild type and single mutants. The Xaa/Pro mutations stabilized the enzyme and did also tend to decrease the catalytic efficiency more than the Ser/Ala mutations.
Arnórsdóttir
Jóhanna
J
Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107, Reykjavík, Iceland.
Helgadóttir
Sunna
S
Thorbjarnardóttir
Sigrídur H
SH
Eggertsson
Gudmundur
G
Kristjánsson
Magnús M
MM
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D003080:Cold Temperature; D004795:Enzyme Stability / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics / Q000378:metabolism; D020860:Subtilisin / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014733:Vibrio / Q000201:enzymology / Q000235:genetics
D000596:Amino Acids; D012694:Serine; D011392:Proline; D020860:Subtilisin; D000409:Alanine