Inhibition kinetics of hydrogen peroxide on β-N-acetyl-d-glucosaminidase from prawn (Penaeus vannamei)

Figures & data

Scheme 1 Kinetic scheme for inactivation of NAGase by H₂O₂.

Figure 1 Determination of the kinetic parameters of prawn NAGase. Conditions were 2 ml system containing 0.15 M NaAc-HAc buffer (pH 5.2) and different concentrations of pNP-β-D-GlcNAc at 37°C. The enzyme final concentration was 0.020 μM. The inset is a Lineweaver-Burk plot for the determination of K_m and V_m for NAGase on the hydrolysis of pNP-β-D-GlcNAc.

Figure 2 Inhibition of the enzyme in different concentration of H₂O₂ solution. Conditions were as same as in Figure 1 except that pNP-β-D-GlcNAc concentration was 0.5 mM with different concentrations of H₂O₂. The enzyme final concentration was 0.020 μM.

Figure 3 Effects of prawn NAGase concentration on its activity at different concentrations of H₂O₂. The concentrations of H₂O₂ for curves 0–5 were 0, 0.2, 0.4, 0.6, 0.8 and 1.0 M, respectively. Assay conditions were same as in Figure 2.

Figure 4 Lineweaver-Burk plots for the enzyme in different concentrations of H₂O₂. The H₂O₂ concentration for lines 0–4 was 0, 0.2, 0.4, 0.6 and 0.8 M, respectively. Conditions were the same as in Figure 1.

Figure 5 Course of inhibition of the enzyme in different concentrations of H₂O₂. The assay conditions were the same as in Figure 1 except that the concentration of pNP-β-D-GlcNAc was 0.4 mM. (a) Substrate reaction course. The final H₂O₂ concentrations for curves 0–4 were 0, 0.2, 0.4, 0.6, and 0.8 M, respectively. (b) Semilogarithmic plots of ln([P]_calc − [P]_t) against time. Data were taken from curves 1–4 in (a).

Figure 6 Course of inhibition at different substrate concentration in the presence of 0.40 M H₂O₂. (a) Curves 1–5 are progress curves with 0.50, 0.40, 0.33, 0.25 and 0.20 mM pNP-β-D-GlcNAc, respectively. (b) Semilogrithmic plot of ln ([P]_calc − [P]_t) against time. Data were taken from curves 1–5 in (a).

Figure 7 Secondary plots of the slopes of the semilogarithmic plots versus [H₂O₂] for a series of fixed substrate concentrations. The data for curve 2 is from Figure 5b (at 0.40 mM substrate). Substrate concentrations for curves 1–5 were 0.50, 0.40, 0.33, 0.25, 0.20 mM, respectively.

Table I.  Microscopic rate constants of the inactivation of prawn NAGase in H₂O₂ solution.

Enzyme forms	Rate constants
Native enzyme	k+0	2.9 × 10^− 3 M^− 1 s^− 1
	k− 0	1.45 × 10^− 3 s^− 1
Enzyme-substrate complex		0.48 × 10^− 3 M^− 1 s^− 1
		1.45 × 10^− 3 s^− 1

Figure 8 Plot of A/v versus 1/[S]. The values of v and A were obtained from Figures 1 and 7.

Figure 9 Fluorescence emission spectra of the enzyme inactivated by H₂O₂. The final concentration of the enzyme was 0.475 μM. The H₂O₂ concentration for curves 1–8 were 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.9 and 1.2 M, respectively.