Purification, Characterization, and Effect of Thiol Compounds on Activity of the Erwinia carotovora L-Asparaginase

Abstract

L-asparaginase was extracted from Erwinia carotovora and purified by ammonium sulfate fractionation (60-70%), Sephadex G-100, CM cellulose, and DEAE sephadex chromatography. The apparent Mr of enzyme under nondenaturing and denaturing conditions was 150 kDa and 37 ± 0.5 kDa, respectively. L-asparaginase activity was studied in presence of thiols, namely, L-cystine (Cys), L-methionine (Met), N-acetyl cysteine (NAC), and reduced glutathione (GSH). Kinetic parameters in presence of thiols (10-400 μM) showed an increase in V(max) values (2000, 2223, 2380, 2500, and control 1666.7 μmoles mg(-1)min(-1)) and a decrease in K(m) values (0.086, 0.076, 0.062, 0.055 and control 0.098 mM) indicating nonessential mode of activation. K(A) values displayed propensity to bind thiols. A decrease in V(max)/K(m) ratio in concentration plots showed inverse relationship between free thiol groups (NAC and GSH) and bound thiol group (Cys and Met). Enzyme activity was enhanced in presence of thiol protecting reagents like dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and GSH, but inhibited by p-chloromercurybenzoate (PCMB) and iodoacetamide (IA).

Figure 1

Purification of L-asparaginase from the E. carotovora. (a) CM cellulose chromatography of the active fractions collected from the Sephadex G-100 gel filtration column. (b) DEAE cellulose Anion Exchange Chromatography of the active fractions collected from the CM cellulose chromatography column.

Figure 2

(a) Native PAGE of the purified L-asparaginase from the E. carotovora, (i) molecular weight marker proteins, (ii) purified asparaginase 10 μgm/mL, (iii) purified asparaginase 25 μgm/mL. (b) SDS-PAGE of the purified L-asparaginase from the E. carotovora, lane 1 molecular weight marker proteins, lane 2 purified asparaginase after DEAE sephadex chromatography, lane 3 purified asparaginase after CM Cellulose chromatography, lane 4 crude preparation.

Figure 3

Calibration curve for the determination of molecular weight of L-asparaginase by gel filtration chromatography.

Figure 4

Lineweaver-Burk analyses of activity shown by asparaginase from E. carotovora in the presence of different concentrations of Cys (a), Met (b), NAC (c), and GSH (d).

Figure 5

The secondary replots of 1/Δ slope versus 1/[Cys] (a), 1/Δ slope versus 1/[Met] (b), 1/Δ slope versus 1/[NAC] (c), and 1/Δ slope versus 1/[GSH] (d).

Figure 6

Effect of thiol containing compounds on the relative V_max/K_m ratio of L-asparaginase from E. carotovora.

Scheme 1

Model for the nonessential activation of asparaginase by thiol containing compounds.

Scheme 2

Acyl enzyme intermediate attack by nucleophile Tyr/Thr. The probable acyl enzyme intermediate of asparaginase from E. coli where Thr is a nucleophile in the N-terminal position.