Azide anions inhibit GH-18 endochitinase and GH-20 Exo β-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.
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.
Biochemistry-Electrochemistry Research Unit, School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima, Thailand and.
Paknisa
P
Sirimontree
Department of Advanced Biosciences, Kinki University, Nara, Japan.
Tamo
T
Fukamizo
Biochemistry-Electrochemistry Research Unit, School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima, Thailand and wipa@sut.ac.th.
Wipa
W
Suginta
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000838:Anions / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D002384:Catalysis; D020134:Catalytic Domain / Q000187:drug effects; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D002688:Chitinases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D048271:Chitosan; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides; D011485:Protein Binding; D014733:Vibrio / Q000201:enzymology*
D000838:Anions; D001386:Azides; D001426:Bacterial Proteins; D009844:Oligosaccharides; C493484:oligochitosan; D002686:Chitin; D048271:Chitosan; D002688:Chitinases; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine
chitin turnover; family 18 chitinase; family 20 N-acetylglucosaminidase; reversible inhibition; sodium azide