Sirimontree Paknisa, Fukamizo Tamo, Suginta Wipa
Biochemistry-Electrochemistry Research Unit, School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima, Thailand and.
Department of Advanced Biosciences, Kinki University, Nara, Japan.
J Biochem. 2016 Feb;159(2):191-200. doi: 10.1093/jb/mvv087. Epub 2015 Sep 1.
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.
哈维氏弧菌是一种发光海洋细菌，它利用几丁质作为唯一能量来源。在几丁质降解过程中，该细菌主要分泌一种内切几丁质酶A（VhChiA）来水解几丁质，产生几丁寡糖片段，这些片段很容易被转运到细胞内，并被一种外切β-N-乙酰氨基葡萄糖苷酶（VhGlcNAcase）分解为N-乙酰葡糖胺单体。在此我们报告，钠盐，尤其是叠氮化钠，以不同的作用方式抑制这两类几丁质降解酶（VhChiA和VhGlcNAcase）。对pNP-糖苷底物酶促水解的动力学分析表明，叠氮化钠对VhChiA的抑制作用具有混合型模式，但对VhGlcNAcase的抑制作用具有竞争性。我们提出，叠氮阴离子通过作为强亲核试剂攻击催化性谷氨酸的Cγ或相邻天冬氨酸残基的Cβ来抑制几丁质酶活性。叠氮阴离子不仅可能与催化中心结合，还可能与VhChiA底物结合裂隙中的其他亚位点结合。相比之下，叠氮阴离子可能仅仅占据VhGlcNAcase的小结合口袋，从而通过短链底物阻断其活性位点的可及性。
