Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414.
beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.
Estrada
P
P
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Mata
I
I
Dominguez
J M
JM
Castillón
M P
MP
Acebal
C
C
D001592:Benzyl Alcohols / Q000378:metabolism / Q000494:pharmacology; D002475:Cellobiose / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005959:Glucosidases / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D003904:Mitosporic Fungi / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D001592:Benzyl Alcohols; D005960:Glucosides; D009596:Nitrophenols; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; C005696:salicin; D008320:Maltose; D005959:Glucosidases; D001617:beta-Glucosidase; D005947:Glucose; C024836:4-nitrophenol