The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

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Abstract
The cellulases cellobiohydrolase 1 (CBH 1) and endoglucanase 1 (EG 1) from the fungus Trichoderma reesei are closely related with 40% sequence identity and very similar in structure. In CBH 1 the active site is enclosed by long loops and some antiparallel β-strands forming a 40 Å long tunnel, whereas in EG 1 part of those loops are missing so that the enzyme has a more common active site groove. Both enzymes were immobilized on silica and these materials were used as chiral stationary phases for chromatographic separation of the enantiomers of two chiral drugs, propranolol and alprenolol. The CBH 1 phase showed much better resolution than did the EG 1 phase, suggesting that the tunnel structure of the protein may play an important role in the chiral separation. The chiral compounds were found to be competitive inhibitors of both enzymes when p-nitrophenyl lactoside (pNPL) was used as substrate. (S)-enantiomers showed stronger inhibitory effects and also longer retention time on the stationary phases than the (R)-enantiomers. The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.