Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS.
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 alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-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 approximately 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.
School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Wipa
W
Suginta
Archara
A
Vongsuwan
Chomphunuch
C
Songsiriritthigul
Jisnuson
J
Svasti
Heino
H
Prinz
D001665:Binding Sites; D002384:Catalysis; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006868:Hydrolysis; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases