The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants.
The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cellobiose or beta-xylobiose. It uses a double-displacement mechanism involving a glycosyl-enzyme intermediate which is formed and hydrolyzed with general acid/base catalytic assistance. Glu127 was proposed as the acid/base catalyst on the basis of sequence alignments, and mutants at this position were constructed in which the glutamic acid is replaced by alanine or glycine. The following kinetic analysis provides firm support for the assignment of Glu127 as the acid/base catalyst and suggests a more general strategy for identification of this residue in other glycosidases. Substrates which do not require protonic assistance for initial bond cleavage exhibit kcat/Km values similar to those of wild-type enzyme, whereas substrates which do require assistance have kcat/Km values over 6000-fold smaller. Thus rate constants for glycosylation are affected to different degrees by this substitution, depending upon their need for acid catalysis. The deglycosylation rate constant is decreased 200-fold by such substitution, due to the removal of general base catalytic assistance. In the presence of sodium azide a new product, beta-cellobiosyl azide, is formed with these mutants whereas only cellobiose is formed with wild-type enzyme or the Glu127Asp mutant under similar conditions. Addition of azide results in very significant increases in kcat values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitrophenyl cellobioside, whereas kcat/Km values for these substrates remain essentially constant. No effects on rate upon azide addition are seen with substrates containing aglycons of poor leaving group ability.(ABSTRACT TRUNCATED AT 250 WORDS)
MacLeod
A M
AM
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Lindhorst
T
T
Withers
S G
SG
Warren
R A
RA
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001483:Base Sequence; D002384:Catalysis; D002475:Cellobiose / Q000378:metabolism; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D044643:Glucan 1,4-beta-Glucosidase; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D019810:Sodium Azide; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D004187:Disaccharides; D005971:Glutamates; D002475:Cellobiose; D018698:Glutamic Acid; D019810:Sodium Azide; D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase; C004173:xylobiose