Inactivation of jack bean urease by N-ethylmaleimide: pH dependence, reversibility and thiols influence.
N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 degrees C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.
Kot
Mirosława
M
Faculty of Chemistry, Jagiellonian University, Kraków, Poland. kot@chemia.uj.edu.pl
Bicz
Anna
A
D032449:Canavalia / Q000201:enzymology*; D004229:Dithiothreitol / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014510:Urease / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D014510:Urease; D005033:Ethylmaleimide; D004229:Dithiothreitol