INHIBITION OF ESCHERICHIA-COLI BETA-GALACTOSIDASE BY 2-NITRO-1-(4,5-DIMETHOXY-2-NITROPHENYL) ETHYL, A PHOTOREVERSIBLE THIOL LABEL

1-Nitro-2-phenylethene (beta-nitrostyrene, 1), which is a thiol-protec ting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. I nt. Ed. Engl. 14, 817-818), was demonstrated in this work to be an irr eversible inhibitor of beta-galactosidase (EC 3.2.1.23), an enzyme kno wn to be inhibited by some thiol reagents or through modifying a methi onine residue at the active site. No reversal of the inhibition was ob served upon subsequent incubation with mercaptoethanol or irradiation (350 nm), 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene (2) was also s hown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the e nzyme. K-cat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-glaacctoyranoside (ONPG) as the substrate and at the highest i nhibitor concentrations employed for compound 1 (4.06 . 10(-4) M) rang ed from 1.67 . 10(4) s(-1) after 30 min of preincubation to < 0.07 . 1 0(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 preincu bation to 1.15 . 10(4) s(-1) after 180 min of preincubation; the chang es in K-m(app), however, were small. The activity was not recovered fo llowing incubation with mercaptoethanol. Since compound 2 and the inhi bited enzyme are 2-nitrobenzyl derivatives, they are expected to be ph otosensitive and indeed, irradiation of the inhibited enzyme in the pr esence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the en zyme activity.