Hydrolyses of alpha- and beta-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

We have measured the hydrolyses of alpha- and beta-cellobiosyl fluorides by the Ce16A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Va rrot, Hastrup, Schulein and Davies (1999) Biochem. J. 337, 297-304]. The be ta-fluoride is hydrolysed according to Michaelis-Menten kinetics by both en zymes. When the similar to 2.0 % of beta-fluoride which is an inevitable co ntaminant in all preparations of the alpha-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A e nzymes show a sigmoidal dependence of rate on substrate concentration, as w ell as activation by cellobiose. These kinetics are consistent with the cla ssic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydro lysis of the 'wrong' glycosyl fluoride for both enzymes. The Michaelis-Ment en kinetics of alpha-cellobiosyl fluoride hydrolysis by the T. reesei enzym e, and its inhibition by cellobiose, previously reported [Konstantinidis, M arsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. H-1 NMR monitoring of the hydrolysis of alpha-cellobiosyl fluoride by both enzymes reveals that in neither case is alpha-cellobiosyl fluoride released into so lution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.