Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 1. .beta.-Galactosidase-catalyzed hydrolysis

Abstract

Oxygen-18 leaving group kinetic isotope effects (KIE) were determined on both Vmax (V) and Vmax/Km (V/K) for the Escherichia coli .beta.-galactosidase-catalyzed hydrolysis of P-nitrophenyl .beta.-D-galactoside (I) and 2,4-dinitrophenyl .beta.-D-galactoside (II). The former substrate exhibits KIE of 1.022 .+-. 0.002 and 1.014 .+-. 0.003 on V and V/K, respectively, while corresponding KIE for the latter are 1.002 .+-. 0.009 and 1.030 .+-. 0.003. Bond scission is largely rate determining for I but not for II at substrate saturation. The 1st irreversible step for both substrates must involve cleavage of the bond to the nitrophenyl leaving group. The mechanism proposed for this reaction is characterized by 2 parallel pathways for substrate hydrolysis. The predominant route for all but the most reactive substrates involves a SN2 nucleophilic displacement of aglycon by the enzyme to yield a covalent galactosyl-enzyme which in turn is hydrolyzed via a nucleophilic attack by water. The most reactive substrates (e.g., II) form transiently an enzyme-bound galactosyl oxocarbonium ion which partitions between enzymes to give the covalent galactosyl-enzyme and H2O to yield galactose.