The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate. |
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Authors: | A L Fink K J Angelides |
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Affiliation: | Division of Natural Sciences, University of California, Santa Cruz, California 95064 USA |
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Abstract: | The reaction of β-galactosidase ( K12) with -nitrophenyl-β--galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of -nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with Ea = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on Km but caused a small decrease in Kcat. |
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