Chemical modification of beta-glucosidase from Trichoderma reesei QM 9414.
The inhibition of beta-glucosidase from Trichoderma reesei QM 9414 by several specific reagents was studied. Diethylpyrocarbonate (DEP) nearly abolished the enzyme activity at concentrations above 10 mM. The presence of substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.02 mM-1.min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.2. Difference spectra at 242 nm and the reversal of the inactivation in the presence of 1 M hydroxylamine indicated the modification of histidine residues. Statistical analysis of residual fractional activity versus the number of modified histidine residues indicated that one histidine residue is essential for catalysis. p-Hydroxymercuribenzoate completely inhibited the enzyme at concentrations of the reagent above 2 mM. Substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.002 mM-1.min-1. Treatment of the modified enzyme with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) showed that one cysteine residue was essential for activity. At pH 5.0 2-ethoxy-1-ethoxy-carbonyl-1,2-dihydroquinoline (EEDQ) inactivated the enzyme according to pseudo-first order kinetics with a second-order rate constant of 0.12 min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.64, indicating the modification of a carboxyl group essential for activity.
de la Mata
I
I
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain.
Castillón
M P
MP
Domínguez
J M
JM
Macarrón
R
R
Acebal
C
C
D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology*; D007700:Kinetics; D011804:Quinolines / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D014242:Trichoderma / Q000201:enzymology*; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D006902:Hydroxymercuribenzoates; D011804:Quinolines; C030601:4-hydroxymercuribenzoate; D006639:Histidine; C000470:EEDQ; D004228:Dithionitrobenzoic Acid; D001617:beta-Glucosidase; D004047:Diethyl Pyrocarbonate