Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl, a photoreversible thiol label

Abstract

1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817–818), was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or though modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene ( 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. K cat values of β-galactosidase at pH 8.3 with o-nitrophenyl β- d-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 · 10 −4 M) ranged from 1.67 · 10 4 s −1 after 30 min of preincubation to < 0.07 · 10 4 s −1 after 180 min preincubation. For compound 2 (9.5 · 10 −5 M) K cat values ranged from 2.70 · 10 4 s −1 following 30 min preincubation to 1.15 · 10 4 s −1 after 180 min of preincubation; the changes in K m app, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.