Inhibition of jack bean urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the inhibition mechanism.
1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of Ki = 0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of Ki* = 4.5 x 10(-5) mM. The respective inhibition constants for DMBQ were Ki = 0.42 mM, Ki* = 1.2 x 10(-3) mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 x 10(-5) mM for BQ and 0.98 x 10(-3) mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.
Zaborska
Wiesława
W
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland.
Kot
Mirosława
M
Superata
Kinga
K
D016227:Benzoquinones / Q000737:chemistry / Q000494:pharmacology*; D053138:Cyclohexenes; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D014510:Urease / Q000037:antagonists & inhibitors*
D016227:Benzoquinones; D053138:Cyclohexenes; D004791:Enzyme Inhibitors; C020617:2,5-dimethyl-4-benzoquinone; D014510:Urease; D003545:Cysteine