Improving the thermostability of Bacillus stearothermophilus neutral protease by introducing proline into the active site helix.
A proline residue was introduced into the N-terminus (Ile140 and Asp141), the middle (Leu147) and the C-terminus (Asp153) of the active site helix of Bacillus stearothermophilus neutral protease for comparing the effects on the thermostability. Introduction of a proline residue into the N-terminus at sites 140 and 141 increased the half-survival temperature (HST) by 7.5 and 2.8 degrees C, respectively, from 68.3 degrees C of the wild-type enzyme. A proline residue at Leu147 decreased the HST by 10.2 degrees C, while no change was observed by introducing a proline residue in the C-terminus. These results were coincidental with the CD data which indicated increases in Tm values of 4.4 and 2.3 degrees C for I140P and D141P, respectively. Susceptibility to alpha-chymotrypsin hydrolysis markedly decreased in mutants I140P and D141P, while increasing in L147P. Molecular modeling suggested that glycine residues on the N-terminus side of proline residues in I140P and D141P relaxed the possible strain caused by proline introduction. The thermostability can, therefore, be explained based on changes in the molecular rigidity.
Nakamura
S
S
Department of Food Science, The University of British Columbia, Vancouver, Canada.
Tanaka
T
T
Yada
R Y
RY
Nakai
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003198:Computer Simulation; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011392:Proline; D013329:Structure-Activity Relationship
D011392:Proline; D010450:Endopeptidases; D002918:Chymotrypsin