Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.
In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.
Krajewska
Barbara
B
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland. krajewsk@chemia.uj.edu.pl
Zaborska
Wiesława
W
D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D001888:Boric Acids / Q000737:chemistry; D032449:Canavalia / Q000201:enzymology*; D002625:Chemistry, Organic / Q000379:methods; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008956:Models, Chemical; D008968:Molecular Conformation; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014510:Urease / Q000737:chemistry*
D001888:Boric Acids; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D013438:Sulfhydryl Compounds; C006358:acetohydroxamic acid; D004228:Dithionitrobenzoic Acid; D014510:Urease; C032688:boric acid