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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/16323
Title: Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS
Authors: Wipa Suginta
Archara Vongsuwan
Chomphunuch Songsiriritthigul
Jisnuson Svasti
Heino Prinz
Suranaree University of Technology
National Synchrotron Research Center, Thailand
Mahidol University
Max Planck Institut fur molekulare Physiologie
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jul-2005
Citation: FEBS Journal. Vol.272, No.13 (2005), 3376-3386
Abstract: The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of α and β anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated β-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed ≈20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme. © 2005 FEBS.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=22144480668&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/16323
ISSN: 1742464X
Appears in Collections:Scopus 2001-2005

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