The determinants of alpha-amylase pH-activity profiles.
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha-amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 alpha-amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the p K(a) values of the active site residues. More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha-amylases.
Nielsen
J E
JE
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany. jnielsen@mccammon.ucsd.edu
Borchert
T V
TV
Vriend
G
G
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001407:Bacillus / Q000201:enzymology; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D014644:Genetic Variation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D011522:Protons; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases