The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides

Abstract

1. Steady-state kinetic parameters for the β-galactosidase-catalysed hydrolysis of 13 aryl β-d-galactopyranosides show no simple dependence on aglycone acidity. 2. α-Deuterium kinetic isotope effects (kH/kD) for seven of these substrates, measured under steady-state conditions with [S]»Km, vary from 1.00 for poor substrates to 1.25 for hydrolysis of the galactosyl-enzyme. 3. Methanolysis of the galactosyl-enzyme in 1.5m-methanol increases KH/kD for degalactosylation, but leaves that for hydrolysis of ‘slow’ substrates unchanged. 4. These data are incompatible with a simple two-step mechanism. A scheme consisting of a conformation change, liberation of a galactopyranosyl cation in an intimate ion-pair, non-productive but preferential collapse of the ion-pair to a covalent species and reaction of the galactosyl enzyme through the ion-paired form is proposed. 5. This scheme is used to rationalize previously puzzling data about the enzyme mechanism.