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 Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The beta-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the approximately 2.0% of beta-fluoride which is an inevitable contaminant in all preparations of the alpha-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei 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 alpha-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. (1)H NMR monitoring of the hydrolysis of alpha-cellobiosyl fluoride by both enzymes reveals that in neither case is alpha-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.
Becker
D
D
Department of Paper Science, UMIST, Sackville Street, Manchester M60 1QD, U.K.
Johnson
K S
KS
Koivula
A
A
Schülein
M
M
Sinnott
M L
ML
D000494:Allosteric Regulation; D002475:Cellobiose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D006868:Hydrolysis / Q000187:drug effects; D003904:Mitosporic Fungi / Q000201:enzymology*; D008956:Models, Chemical; D013237:Stereoisomerism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
C074099:cellobiosyl fluoride; D002475:Cellobiose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase