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

被引:12
作者
Becker, D
Johnson, KSH
Koivula, A
Schülein, M
Sinnott, ML
机构
[1] Univ Manchester, Dept Paper Sci, Manchester M60 1QD, Lancs, England
[2] VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
[3] Novo Nordisk AS, DK-2880 Bagsvaerd, Denmark
关键词
co-operativity; Hehre mechanism; transglycosylation;
D O I
10.1042/0264-6021:3450315
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 [Varrot, Hastrup, Schulein and Davies (1999) Biochem. J. 337, 297-304]. The beta-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the similar to 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 J. 291, 883-888] are withdrawn. 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 solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.
引用
收藏
页码:315 / 319
页数:5
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