Effects of both shortening and lengthening the active site nucleophile of Bacillus circulans xylanase on catalytic activity.
The relative positioning of the two carboxyl groups at the active site of glycosidases is crucial to their function and the mechanism followed. The distance between these two groups in Bacillus circulans xylanase has been modified by mutagenesis of the catalytic nucleophile Glu78. An increase in the separation (Glu78Asp) results in a large (1600-5000-fold) reduction in the rate of the glycosylation step, but little change in the extent of bond cleavage or proton donation at the transition state. A decrease in the separation was achieved by selective carboxymethylation of the Glu78Cys mutant. This modified mutant was only 16-100-fold less active than wild-type enzyme, and its transition state structure was similarly unchanged. Complete removal of the carboxyl group (Glu78Cys) resulted in a mutant with no measurable catalytic activity. Furthermore, it did not even undergo the first step, glycosylation of the active site thiol. These results confirm the importance of precise positioning of the nucleophile at the active site of these enzymes.
Lawson
S L
SL
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Wakarchuk
W W
WW
Withers
S G
SG
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D003545:Cysteine; D018698:Glutamic Acid; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism*
D007461:Iodoacetates; D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine; D019807:Iodoacetic Acid