Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process.
p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.
Kot
Mirosława
M
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland. kot@chemia.uj.edu.pl
D016227:Benzoquinones / Q000494:pharmacology*; D032449:Canavalia / Q000145:classification / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014510:Urease / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; C004532:quinone; D014510:Urease