title: Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label.
abstract: 1-Nitro-2-phenylethene (beta-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 beta-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through 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. Kcat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 x 10(-4) M) ranged from 1.67 x 10(4) S-1 after 30 min of preincubation to <0.07 x 10(4) S-1 after 180 min preincubation. For compound 2 (9.5 x 10(-5) M) Kcat values ranged from 2.70 x 10(4) S-1 following 30 min preincubation to 1.15 x 10(4) S-1 after 180 min of preincubation; the changes in Km(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.
authors.affiliation: Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
authors.forename: R
authors.initials: R
authors.lastname: Golan
authors.forename: U
authors.initials: U
authors.lastname: Zehavi
authors.forename: M
authors.initials: M
authors.lastname: Naim
authors.forename: A
authors.initials: A
authors.lastname: Patchornik
authors.forename: P
authors.initials: P
authors.lastname: Smirnoff
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008956:Models, Chemical; D015394:Molecular Structure; D009578:Nitrobenzenes / Q000494:pharmacology*; D010782:Photolysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors*
chemical_list: C099265:2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethene; D004791:Enzyme Inhibitors; D009578:Nitrobenzenes; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; D001616:beta-Galactosidase