Suicide-substrate inactivation of β-galactosidase by diazomethyl β-d-galactopyranosyl ketone

Abstract Diazomethyl β- d -galactopyranosyl ketone ( 1 ) has been proven to be a mechanism-based, irreversible (suicide-substrate) inactivator of Aspergillus oryzae β- d -galactosidase, but not an inactivator of E. coli lacZ β- d -galactosidase. Compound 1 is stable in buffers of normal physiological pH. It is decomposed by H + , but not by nucleophiles. Inactivation of A . oryzae β- d -galactosidase was proven to be mechanism-based and irreversible via experiments which showed that the enzyme could be protected from inactivation by a competitive inhibitor, neither diazomethyl β- d -glucopyranosyl ketone ( 2 ) nor diazomethyl α- d -galactopyranosyl ketone inactivated the enzyme and therefore inactivation is stereospecific, excess inhibitor could be separated from inactive enzyme without regain of activity and therefore it is bound irreversibly, and a second pulse of enzyme is inactivated at the same rate as enzyme inactivated to 95% activity by the first pulse. Diazomethyl β- d -glucopyranosyl ketone ( 2 ) inhibited sweet almond β- d -glucosidase.