|Title||THE ACID/BASE CATALYST IN THE EXOGLUCANASE/XYLANASE FROM CELLULOMONAS-FIMI IS GLUTAMIC-ACID-127 - EVIDENCE FROM DETAILED KINETIC-STUDIES OF MUTANTS|
|Publication Type||Journal Article|
|Year of Publication||1994|
|Authors||MACLEOD, AM, LINDHORST, T, Withers, SG, WARREN, RAJ|
|Date Published||MAY 24|
The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cerlobiose 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 k(cat)/K-m values similar to those of wild-type enzyme, whereas substrates which do require assistance have k(cat)/K-m 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 Glu 127Asp mutant under similar conditions. Addition of azide results in very significant increases in k(cat) values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitropheny1 cellobioside, whereas k(cat)/K-m 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. These results suggest that azide occupies a vacant anionic site created by removal of the acid/base catalyst and reacts rapidly with the glycosyl-enzyme intermediate, increasing the steady-state rate and forming the glycosyl azide product. The techniques employed in this study may be generally applicable to the identification of the acid/base catalyst in any cloned glycosidases belonging to sequence-related families.