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

摘要

pWe have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of iHumicola insolens/i and iTrichoderma reesei/i, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. b337/b, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of iT. reesei/i before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the iT. reesei/i enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. b291/b, 883-888] are withdrawn. sup1/supH NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed./p