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ENZYMATIC CATALYSIS IN CRYSTALS
OF E.COLI MALTODEXTRIN PHOSPHORYLASE
Mara Campagnolo,
Dipartimento di Scienze Chimiche, Università di Trieste.
Maltodextrin Phosphorylase (MalP) is a bacterial homodimeric enzyme of
776 amino acids (Fig. 1) that catalyses the phosphorylysis of an
a-1,4-glycosidic bond from the non-reducing end of linear
oligosaccharide (maltodextrin), to yield glucose-1-phosphate (G1P).
MalP is an isoenzyme of the mammalian Glycogen Phopshorylase (GP), with
44% of identity in aminoacidic sequence and the catalytic site with
100% identity. MalP is a simpler enzyme than GP since it is not an
allosteric protein and it is already active in its native form.
Furthermore, it has higher affinity for linear sugars than GP. The
crystal structures of oligosaccharide bound across the catalytic site
of MalP both binary and ternary enzyme-substrate complexes has been
recently obtained.1 In the CEB's laboratory of Trieste (Italy) we have
determined the structure of the binary complex of MalP with G1P, the
product of phosphorylation on oligosaccharides, by co-crystallization
experiments with the hanging-drop vapor diffusion method. X-ray
diffraction experiments on this crystal were carried out at the Elettra
Synchrotron Light Source of Trieste. A complete data set of the
MalP-G1P complex has been collected to 2.0 Å of resolution.2 The
electronic density map of the catalytic site of the binary complex
MalP-G1P (Figure 2) clearly reveals that the catalytic site is fully
occupied by the substrate.
Figure 1. Dimeric structure
of MalP-oligosaccharidic complex.
Figure 2. The electronic
density map of the catalytic site in the binary complex MalP-G1P.
e of the PLP cofactorof G1P is located close to the negative charged
5'-phosphat--More--(60%) but it does not form direct H-bond, while, it
is form two strong H-bonds with the positive charged Arg569. In the
constitutively active MalP this positively charged residue can
stabilize the negative charged G1P, whereas in the inactive form of GP
the Arg569 is held away from the catalytic site by the loop 280
involved in the allosteric transition of the mammalian enzyme.
Crystallographic studies on complexes of MalP revealed an important
loop (loop 380) whose role is associated with the binding of natural
oligosaccharidic substrates. This loop has been found into two
different conformation: open and close. In the structures of MalP
complexes with linear natural substrates the loop380 was found in a
close conformation. In the binary complex MalP-G1P we have found it in
the close conformation suitable for the specific recognition of an
oligosaccharidic substrate. For this reason we used crystals of this
binary complex for studies of enzymatic catalysis in solid state, by
soaking with oligosaccharidic substrates maltotetraose (G4) and
maltopentaose (G5). The electronic density map of the catalytic site of
binary hosphorylation of G1P. clearly shows the presence of phosphate,
coming from dep--More--(67%) In the catalytic channel instead of the
expected five glucosidic units coming from the elongated chain we have
localised only four glucosidic monomers. Also the map of the catalytic
site of the structure resulting from the soaking with G5 reveals the
phosphate and only five of the six glucosidic units. These experimental
results seem not confirm the elongation of the chain, as should be
expected from the experiment of dephosphorylation. Nevertheless, in
both structures, the configuration of the anomeric carbon of the last
external glucosidic unit is found inverted respect to the more stable
b-configuration (Fig. 3)1. From this result it can be conclude that
this carbon is not the reducing end of the oligosaccharidic chain but
there is at least another a-1,4 glycosidic link. The glucosidic unit
that should be located outside the catalytic channel is not detected in
the electronic density map probably for high conformational disorder.
These experiments show that the relative large oligosaccharidic
substrate can diffuse into the whole crystal icant crystal damage. the
enzymatic reaction can occur completely without signif--More--(74%) The
determination of these three-dimensional structures, that represent
several steps of the enzymatic pathway, has allowed to draft a
molecular movie that gives an idea of how the enzyme works.
Figure 3. The electronic
density map of the reducing end of the oligosaccharidic chain in the
catalytic site and the configuration of the anomeric carbon in the
complex of MalP-G1P soaked with G4 (A) and in the complex of MalP-G5
(B)
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1 K. A. Watson, C. Mc Cleverty, S.Geremia, L. N. Johnson,
Phosphorylase recognition and phosphorolysis of its oligosaccharide
substrate: answers to a long outstanding question, The EMBO Journal,
18, 17, 4618, 1999 2 S. Geremia, L. N. Johnson, K. A. Watson, R.
Schinzel, The structure of maltodextrin phosphoryalse with
glucose-1-phosphate, Elettra Highlights 1999-2000, 17-19, 2001
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