TAILIEUCHUNG - Báo cáo khoa học: 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 fromVibrio carchariaehave been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation ofaandbanomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chi-tinase A primarily generated b-anomeric products, indicating that it cata-lyzed hydrolysis through a retaining mechanism. | iFEBS Journal Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae as revealed by HPLC-MS Wipa Suginta1 Archara Vongsuwan1 Chomphunuch Songsiriritthigul1 2 Jisnuson Svasti3 and Heino Prinz4 1 Schoolof Biochemistry Institute of Science Suranaree University of Technology Nakhon Ratchasima Thailand 2 NationalSynchrotron Research Center Nakhon Ratchasima Thailand 3 Department of Biochemistry and Center for Protein Structure and Function Faculty of Science MahidolUniversity Bangkok Thailand 4 Max Planck Institut fur Molekulare Physiologie Dortmund Germany Keywords chitinase A chitooligosaccharides quantitative HPLC-MS transglycosylation Vibrio carchariae Correspondence W. Suginta Schoolof Biochemistry Suranaree University of Technology Nakhon Ratchasima 30000 Thailand Fax 66 44 224185 Tel 66 44 224313 E-mail wipa@ Received 13 January 2005 revised 21 March 2005 accepted 6 May 2005 doi 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 a and b anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated b-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 Glc-NAc 6 GlcNAc 4 GlcNAc 3 and showed no activity towards Glc-NAc 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 .

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