Azide anions inhibit GH-18 endochitinase and GH-20 Exo β-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi

Abstract

Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin as its sole source of energy. In the course of chitin degradation, the bacterium primarily secretes an endochitinase A (VhChiA) to hydrolyze chitin, generating chitooligosaccharide fragments that are readily transported into the cell and broken down to GlcNAc monomers by an exo β-N-acetylglucosaminidase (VhGlcNAcase). Here we report that sodium salts, especially sodium azide, inhibit two classes of these chitin-degrading enzymes (VhChiA and VhGlcNAcase) with distinct modes of action. Kinetic analysis of the enzymatic hydrolysis of pNP-glycoside substrates reveals that sodium azide inhibition of VhChiA has a mixed-type mode, but that it inhibits VhGlcNAcase competitively. We propose that azide anions inhibit chitinase activity by acting as strong nucleophiles that attack Cγ of the catalytic Glu or Cβ of the neighbouring Asp residues. Azide anions may bind not only to the catalytic centre, but also to the other subsites in the substrate-binding cleft of VhChiA. In contrast, azide anions may merely occupy the small-binding pocket of VhGlcNAcase, thereby blocking the accessibility of its active site by short-chain substrates.

Keywords: chitin turnover; family 18 chitinase; family 20 N-acetylglucosaminidase; reversible inhibition; sodium azide.

Fig. 1

Effects of sodium salts on the specific activity of VhChiA (A) and VhGlcNAcase (B). Reaction mixtures contained 1 μg of VhChiA or 3 μg of VhGlcNAcase, 500 μM of pNP-GlcNAc₂ or pNP-GlcNAc, and 2 M sodium azide, sodium formate, sodium acetate, sodium nitrate or sodium chloride in 100 mM potassium acetate, pH 5.5 or 100 mM potassium phosphate, pH 7.5. Release of pNP was determined as described under Materials and Methods section. The error bars represent the standard errors from triplicate experiments.

Fig. 2

Effects of 0-3 M sodium azide on the structural integrity of VhChiA (A) and VhGlcNAcase (B) and of sodium nitrate on the structural integrity of VhGlcNAcase (C), investigated by fluorescence spectroscopy. The emission spectra were collected from 300 to 500 nm upon excitation at 295 nm. Shifts in the maximum emission wavelength and increases in fluorescence intensity occurred as a result of enzyme denaturation.

Fig. 3

Dose-response plots of VhChiA fractional activity as a function of sodium azide concentrations (A) and VhGlcNAcase fractional activity as a function of sodium azide (circles) or sodium nitrate (squares) concentrations (B). The values of IC₅₀ for sodium azide and sodium nitrate on the two enzymes were determined from this graph. Reaction mixtures (100 µl), contained 500 µM of pNP-GlcNAc₂ or pNP-GlcNAc, 1 µg of VhChiA or 3 µg of VhGlcNAcase, and concentrations of sodium azide or sodium nitrate varied from 0 to 4 M in 100 mM sodium phosphate buffer, pH 7.5. Release of pNP was determined as described under Materials and Methods. The error bars represent the standard errors from triplicate experiments.

Fig. 4

Kinetic properties of VhChiA with pNP-GlcNAc₂ as substrate. Reaction mixtures (100 μl), contained 1 µg VhChiA and varied concentrations of pNP-GlcNAc₂ (0–800 µM) with sodium azide 0 M (circles), 0.5 M (squares), 1 M (triangles), 1.5 M (inverted triangles) or 2.0 M (diamonds) in 100 mM sodium phosphate buffer, pH 7.5. Release of pNP was determined as described under Materials and Methods section. Kinetic parameters were obtained from Michaelis-Menten plots (A) and the type of inhibition was assessed from Lineweaver-Burk plots (B). K_i and αK_i values were derived from Dixon plots (C and D). The error bars represent the standard errors from triplicate experiments.

Fig. 5

Kinetic properties of VhGlcNAcase with pNP-GlcNAc as substrate. Reaction mixtures (100 μl), contained 3 µg VhGlcNAcase and pNP-GlcNAc (0–800 µM) with sodium azide or sodium nitrate 0 M (circles), 0.3 M (squares), 0.4 M (triangles), 0.5 M (inverted triangles) or 0.6 M (diamonds) in 100 mM sodium phosphate buffer, pH 7.5. Release of pNP was determined as described under Materials and Methods. Kinetic parameters of VhGlcNAcase with sodium azide and sodium nitrate were obtained from Michaelis-Menten plots (A) and (B), respectively. Types of inhibition of sodium azide (C) and sodium nitrate (D) were assessed from Lineweaver-Burk plots. K_i values of sodium azide and sodium nitrate derived from Dixon plots (E and F, respectively). The error bars represent the standard errors from triplicate experiments.

Fig. 6

Proposed mechanism of inhibition of the hydrolytic activity of wild-type VhChiA by azide anion.

Scheme I

The mechanism of mixed-type inhibition.

Scheme II

The mechanism of competitive inhibition.