The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease.
The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent to which mutational effects were interdependent. The effects of single point mutations on the k(cat)/K(m) were non-additive, even in cases where the point mutations were located 10 A or more from the active site Zn(2+) and separated from each other by up to 25 A. This shows that catalysis is affected by large electrostatic networks that involve major parts of the enzyme. The interdependence of mutations at positions as much as 25 A apart in space also indicates that other effects, such as active site dynamics, play an important role in determining active site electrostatics. Several mutations yielded a significant increase in the activity, the most active (quadruple) mutant being almost four times as active as the wild type. In some cases the shape of the pH-activity profile was changed significantly. Remarkably, large changes in the pH-optimum were not observed.
de Kreij
Arno
A
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
van den Burg
Bertus
B
Venema
Gerard
G
Vriend
Gert
G
Eijsink
Vincent G H
VG
Nielsen
Jens E
JE
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013816:Thermodynamics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases