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Carboxy‐group modification: high‐temperature activation of charge‐neutralized and charge‐reversed β‐glucosidases from Aspergillus niger
Author(s) -
Rashid Muhammad Hamid,
Siddiqui Khawar Sohail
Publication year - 1998
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1998.tb00499.x
Subject(s) - chemistry , aspergillus niger , stereochemistry , enzyme , glucosidases , nucleophile , reaction rate constant , chemical modification , enzyme kinetics , medicinal chemistry , nuclear chemistry , kinetics , active site , catalysis , biochemistry , physics , quantum mechanics
Purified β‐glucosidase from Aspergillus niger NIAB280 was chemically modified by 1‐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. β‐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 × 10 −2 mM min −1 . Glucose, as competitive inhibitor, partly protected the active‐site carboxy group against chemical modification, with a K d 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 p K a values of the acidic and basic limbs of the native enzyme were 2.9 and 6.5 respectively. β‐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 β‐glucosidase, GAM and EDAM had V max / K m values of 0.73, 1.22 and 0.60 respectively at 40°C. Interestingly, the activation energy profiles of native β‐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 β‐glucosidases at temperatures higher than 50°C. The p K a 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 β‐glucosidase.