The mechanism of action of beta-galactosidase. Effect of aglycone nature and -deuterium substitution on the hydrolysis of aryl galactosides.
1. Steady-state kinetic parameters for the beta-galactosidase-catalysed hydrolysis of 13 aryl beta-d-galactopyranosides show no simple dependence on aglycone acidity. 2. alpha-Deuterium kinetic isotope effects (k(H)/k(D)) for seven of these substrates, measured under steady-state conditions with [S]>>K(m), 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 K(H)/k(D) 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.
D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D005690:Galactose; D005696:Galactosidases; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D000432:Methanol; D008956:Models, Chemical; D008968:Molecular Conformation; D010636:Phenols
D010636:Phenols; D003903:Deuterium; D005696:Galactosidases; D005690:Galactose; D000432:Methanol