Carboxy-group modification: high-temperature activation of charge-neutralized and charge-reversed beta-glucosidases from Aspergillus niger.
Purified beta-glucosidase from Aspergillus niger NIAB280 was chemically modified by l-ethyl-3-(3-dimethyl-aminopropyl)-carbodi-imide (EDC) in the presence of glycinamide (GAM) as nucleophile under various conditions to study the role of carboxy groups in the catalytic mechanism of this enzyme. beta-Glucosidase was inactivated by the binding of one mol of EDC per mol of the enzyme with a second-order rate constant of 4.77 x 10(-2) mM min-1. Glucose, as competitive inhibitor, partly protected the active-site carboxy group against chemical modification, with a Kd of 3.64 mM. The pH dependence of chemical modification by EDC showed that first-order rate constants decreased with increasing pH, indicating that the proton donating group is a carboxy group. The pKa values of the acidic and basic limbs of the native enzyme were 2.9 and 6.5 respectively. beta-Glucosidase was modified by EDC in the presence of GAM and ethylenediamine dihydrochloride (EDAM) as nucleophiles for 60 min. The effects of neutralization (GAM) and reversal (EDAM) of the negative charges of surface carboxy groups on the kinetic properties of the enzyme were also studied. Native beta-glucosidase, GAM and EDAM had Vmax/K(m) values of 0.73, 1.22 and 0.60 respectively at 40 degrees C. Interestingly, the activation energy profiles of native beta-glucosidase (103 and 79 kJ/mol) were biphasic, whereas those of GAM (137, 101 and 30 kJ/mol) and EDAM (285, 100 and 29 kJ/mol) were triphasic, indicating significant activation of modified beta-glucosidases at temperatures higher than 50 degrees C. The pKa values of both the active-site carboxy groups as well as the pH optima of GAM and EDAM were also significantly decreased compared with those of the native beta-glucosidase.
National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
M H
MH
Rashid
K S
KS
Siddiqui
D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry*; D004563:Electrochemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016014:Linear Models; D011522:Protons; D001617:beta-Glucosidase / Q000737:chemistry*
D002264:Carboxylic Acids; D011522:Protons; D001617:beta-Glucosidase