NDLI: Structure and Mechanism of an ADP-Glucose Phosphorylase from Arabidopsis thaliana †,‡

Content Provider	PubMed Central
Author	Mccoy, Jason G. Arabshahi, Abolfazl Bitto, Eduard Bingman, Craig A. Ruzicka, Frank J. Frey, Perry A. Phillips, George N.
Abstract	The X-ray crystal structure of the At5g18200.1 protein has been solved to a nominal resolution of 2.30 Å. 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 adenylyltransfer 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 14C between ADP-[14C]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 k cat = 4.1 s−1 and K m-values of 1.4 µM and 83 µM for ADP-[14C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 °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 k cat = 2.7 s−1 and K m-values of 6.9 µM and 90 µM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 °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 solved to 1.83 Å resolution, and shows the AMP-group bonded to His186. The value of K eq in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 °C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl2.
Related Links	http://dx.doi.org/10.1021/bi052232m
Ending Page	3162
Page Count	9
Starting Page	3154
File Format	PDF
ISSN	00062960
e-ISSN	15204995
Journal	Biochemistry
Issue Number	10
Volume Number	45
Language	English
Publisher Date	2006-03-01
Access Restriction	Open
Subject Keyword	Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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