TY - JOUR
T1 - Hydration of Cellobial by Exo and Endo-Type Cellulases
T2 - Evidence for Catalytic Flexibility of Glycosylases
AU - Kanda, Takahisa
AU - Brewer, Curtis F.
AU - Okada, Gentaro
AU - Hehre, Edward J.
PY - 1986/3
Y1 - 1986/3
N2 - New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo (or Avicelase-) type and endo (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the β-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the β-anomer of β-D-glucopyranosyl(l→4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a β-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme. The results with the cellulases add to growing evidence for the view that glycosylases have catalytic groups that are functionally flexible beyond the needs of the principle of microscopic reversibility and, hence, the potential to act upon different substrates by different mechanisms.
AB - New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo (or Avicelase-) type and endo (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the β-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the β-anomer of β-D-glucopyranosyl(l→4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a β-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme. The results with the cellulases add to growing evidence for the view that glycosylases have catalytic groups that are functionally flexible beyond the needs of the principle of microscopic reversibility and, hence, the potential to act upon different substrates by different mechanisms.
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U2 - 10.1021/bi00353a032
DO - 10.1021/bi00353a032
M3 - Article
C2 - 3964662
AN - SCOPUS:0023046611
SN - 0006-2960
VL - 25
SP - 1159
EP - 1165
JO - Biochemistry
JF - Biochemistry
IS - 5
ER -