Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside |
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Authors: | A L Fink N E Good |
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Institution: | Division of Natural Sciences, University of California Santa Cruz, California 95064 USA |
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Abstract: | The reaction of almond β-glucosidase with -nitrophenyl-β--glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of -nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in Km from 1.7×10?3M (0%) to 1.7×10?2M, whereas Vmax is unchanged. The DMSO thus apparently acts as a competitive inhibitor with Ki = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with Ea = 23.0 ± 2.0 kcal/mole. |
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