Biochem. J. (2006) 395
(99–106) (Printed in Great Britain)
Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase
Marc SAURA-VALLS*, Régis FAURɆ, Sergi RAGÀS*, Kathleen PIENS‡, Harry BRUMER‡, Tuula T. TEERI‡, Sylvain COTTAZ†, Hugues DRIGUEZ† and Antoni PLANAS*1
*Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain, †CERMAV-ICMG-FR-CNRS 2607, 38041 Grenoble Cedex 9, France, and ‡School of Biotechnology, Royal Institute of Technology, 106 91 Stockholm, Sweden
Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcb1,4- and X=[Xyla1,6]Glcb1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 °C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.
Key words: assay, capillary electrophoresis, ping-pong mechanism, xyloglucan endotransglycosylase (XET), xyloglucan oligosaccharides (XGOs).
Abbreviations used: ANTS, 8-aminonaphthalene-1,3,6-trisulphonic acid; EOF, electro-osmotic flow; HPCE, high performance capillary electrophoresis; XEH, XG endohydrolase; XET, XG endotransglycosylase; XG, xyloglucan; XGOs, XG oligosaccharides; XGOol, reduced XGO.
1To whom correspondence should be addressed (email antoni.planas@iqs.es).
Received 25 August 2005/22 November 2005; accepted 15 December 2005
Published as BJ Immediate Publication 15 December 2005, doi:10.1042/BJ20051396
The Biochemical Society, London ©2006
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