Region-directed mutagenesis of residues surrounding the active site nucleophile in beta-glucosidase from Agrobacterium faecalis.
The active site nucleophile of the beta-glucosidase of Agrobacterium faecalis has recently been identified by the use of inhibitors. A combination of site-directed and in vitro enzymatic mutagenesis was carried out on the beta-glucosidase to probe the structure of the active site region. Forty-three point mutations were generated at 22 different residues in the region surrounding the active site nucleophile, Glu358. Only five positions were identified which affected enzyme activity indicating that only a few key residues are important to enzyme activity, thus the enzyme can tolerate a number of single residue changes and still function. The importance of Glu358 to enzymatic function has been confirmed and other residues important to enzyme structure or function have been identified.
Trimbur
D E
DE
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Warren
R A
RA
Withers
S G
SG
D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012231:Rhizobium / Q000201:enzymology*; D016415:Sequence Alignment; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D001617:beta-Glucosidase