Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of alpha-helices.
The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0 degrees C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2 degrees C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.
Eijsink
V G
VG
Department of Genetics, University of Groningen, Haren, The Netherlands.
Vriend
G
G
van den Burg
B
B
van der Zee
J R
JR
Venema
G
G
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics
D008666:Metalloendopeptidases; C044194:microbial metalloproteinases