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Title
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Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.
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Authors
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J.G.McCoy,
A.Arabshahi,
E.Bitto,
C.A.Bingman,
F.J.Ruzicka,
P.A.Frey,
G.N.Phillips.
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Ref.
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Biochemistry, 2006,
45,
3154-3162.
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PubMed id
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Abstract
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The X-ray crystal structure of the At5g18200.1 protein has been determined to a
nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like
fold containing two distinct HIT-like motifs. The sequence of At5g18200.1
indicates a distant family relationship to the Escherichia coli galactose-1-P
uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein
confirms this relationship. The At5g18200.1 protein does not demonstrate GalT
activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose
with various phosphates. The best acceptor among those evaluated is phosphate
itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase.
The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and
glucose-1-P in the absence of phosphate. The steady state kinetics of exchange
follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and
K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P,
respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose
with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady
state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90
microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C.
The kinetics are consistent with a double-displacement mechanism that involves a
covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this
intermediate was determined to 1.83 A resolution and shows the AMP group bonded
to His(186). The value of K(eq) in the direction of ADP and glucose-1-P
formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal
ion, and it is 40 in the presence of 1 mM MgCl(2).
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