Multi-step analysis of Hg2+ ion inhibition of jack bean urease.

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).