3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P28 REACTION MEChANISM OF xyLOGLuCAN
ENDOTRANSGLyCOSyLASE (xET) FROM
PeTrOseliNuM CrisPuM ROOTS
SOňA GARAJOVá, DAnA FLODROVá, VLADIMíR
FARKAŠ and eVA STRATILOVá
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská
cesta 9, 845 38 Bratislava, Slovakia
chemsong@savba.sk
Introduction
Plant xyloglucan endotransglycosylases (XETs, EC
2.4.1.207) catalyze the random cleavage of β-1,4-polyglucose
backbone of the donor molecule (xyloglucan, XG) and
the transfer of part of XG carrying the newly created reducing
end to hydroxyl group at C-4 on the non-reducing end of
another XG molecule or XG-derived oligosaccharide (acceptors).
The process of transglycosylation can be described by
Bi Bi reaction mechanism. Generally, there are two possible
models of Bi Bi reaction mechanism: Pin-Pong and Sequential.
In the Ping-Pong mechanism, the cleavage and transfer
of part of polyglycan chain to the acceptor run in two steps.
The first product (represented by the part of polyglycan chain
carrying the original reducing end) is released from the stable
enzyme-substrate complex before the acceptor substrate can
bind. In contrast to the Ping-Pong mechanism, the sequential
mechanism is observed like one step reaction, where a ternary
complex of enzyme with the both substrates is formed.
The products are released at the same time.
The main XET form of parsley roots with the isoelectric
point 4.6 has a broad pH optimum in the region of its stability
(pH 4.5–9.0) with one maximum in acidic (pH 5.8) and
the second one in alkalic (pH 8.8) region 1 . The kinetic analysis
at these two pH optima was performed using radioactive
alditols of XG octasaccharide (XLXGol + XXLGol) as
an acceptor substrate as well as in dependence on the degree
of polymerization (DP) of reducing xyloglucan oligosaccharides
(XGOs). XGOs with DP 7, 8 and 9 were used.
The mechanism of BiBi reaction was suggested from the
nonlinear regression of kinetic data.
Experimental
E x t r a c t i o n o f X T H f r o m P a r s l e y
R o o t s
XET from parsley roots (Petroselinum crispum cv.
Olomoucká dlouhá) was isolated and partially purified as
described previously 1 .
S u b s t r a t e s
Tamarind seed xyloglucan used in this study was from
Dainippon Pharmaceutical Co., Ltd, Osaka, Japan.
XGOs with DP 7–9 were prepared by digestion of tamarind
xyloglucan with Trichoderma cellulase 2 . They were
purified on Biogel P2 column and further fractionated by
preparative HPLC on TSK Gel Amide column (Tosoh) as
described 3 . XG octasaccharide was converted to the corre-
s633
sponding 1-deoxy-1-aminoalditols (glycamines) by reductive
amination 4 . Radioactive alditol of XG octasaccharide
or reducing XGOs with DP 7, 8 and 9 were prepared by their
reduction with na-borotritide [ 3 H]naBH 4 (ICn Radiochemicals,
San Diego, CA) as described earlier 5 .
K i n e t i c P a r a m e t e r s
Standard XET assays were performed at 29 °C utilizing
the radiometric method according to Fry et al. The initial
rates were determined at pH 5.8 and 8.8 using five different
concentrations of xyloglucan (0.5–5 g dm –3 at pH 5.8 and
0.5–1.5 g dm –3 at pH 8.8, respectively) and of radioactive
alditols of XG octasaccharide (0.6–3.1 µM). Further experiments
were all performed at pH 5.8 using the same XG concentration
range as described previously for this pH. Reducing
XGOs with DP 7, 8 and 9 were used in the range of
5–200 µM. The incorporation of each [1– 3 H]-labelled XGOs
was measured by scintillation counting (Liquid Scintillation
Analyzer Tri-Carb 2800TR, PerkinElmer, Illinois, USA) as
described previously (Sulová et al., 1995). The K M values
were calculated from nonlinear regression using Origin 6.0.
Results
XET as a member of glycoside hydrolase family GH16
utilize a double displacement/retaining mechanism of transfer.
This mechanism involves the formation of a covalent
enzyme-substrate intermediate 6 , what is characteristic for
1/v (nmol -1 .min)
1/v (nmol -1 .min)
75000
50000
25000
150000
100000
50000
0
0 0.5 1 1.5 2 2.5
1/XG (g -1 .L)
0
0 0.5 1 1.5 2 2.5
1/XG (g -1 .L)
Fig. 1. Plot of 1/v = f(1/XG) at different concentrations of mixture
of XLXGol + XXLGol: 0.86 µM (▲), 1 µM (�), 1.24 µM (�),
1.54 µM (◊), 2.1 µM (�) and 3.1 µM (Δ). Michaelis parameters
were determined at ph 5.8 (A) and 8.8 (b), respectively
A
B