Kinetics of jack bean urease inhibition by 2,3-dichloro-1,4-naphthoquinone. Elucidation of the mechanism: redox cycling and sulfhydryl arylation

Figures & data

Figure 1.  (a) Inactivation progress curves as a dependence of urease residual activity vs incubation time at pH 7.8, for different DCNQ concentrations. (b) Dependence of urease residual activity vs incubation time in semilogarithmic system. DCNQ concentration is given numerically.

Table 1.  Kinetic constants of urease inactivation by DCNQ: the rate constants of the fast (k_f) and slow phase (k_s) and the slow phase contribution parameter a.

DCNQ μM	kf min^−1	ks min^−1	a %
3	0.031 ± 0.004	0.006 ± 0.0005	70.5
4	0.042 ± 0.005	0.013 ± 0.001	69.4
8	0.068 ± 0.007	0.032 ± 0.003	66.7
12	0.197 ± 0.015	0.064 ± 0.007	66.7
			av. 68.3 ± 1.9

Figure 2.  Urease residual activity as a function of DCNQ concentration for a 20 minute and 60 minute incubation.

Figure 3.  Thiol influence on urease inactivation by DCNQ relative to the control activity. The percent of the enzyme activity in the presence of DCNQ without the thiol is given for comparison. Concentration of the thiols: L-cysteine (L-cys), glutathione (GSH), dithiothreitol (DTT) and DCNQ in the incubation mixture was equal to 0.5 mM and 8 μM, respectively. Enzyme activity was determined after a 10 and 30 minutes incubation.

Figure 4.  Catalase influence on urease inactivation by DCNQ. DCNQ concentration was equal to 5.5 and 11 μM.

Table 2.  Estimation of hydrogen peroxide production by 7 μM DCNQ in 20 mM phosphate buffer pH 7.8, after a 60 min incubation.

Experimental conditions	H2O2 mM
7 μM DCNQ	~ 0.1
7 μM DCNQ + 0.1 mM DTT	~ 0.4
7 μM DCNQ + 0.2 mM DTT	~ 0.7
7 μM DCNQ + ureaza (1 mg cm^−3)	~ 0.7

Figure 5.  Reactivation of DCNQ inactivated urease by DTT addition. Activity of urease inactivated by DCNQ (•) and after adding DTT (◊). Urease was inactivated by 12 μM DCNQ, in fiftieth minute of the incubation a small aliquot of DTT was added. DTT concentration in the system was equal to 125 μM.