Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.
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).
McCoy
Jason G
JG
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Arabshahi
Abolfazl
A
Bitto
Eduard
E
Bingman
Craig A
CA
Ruzicka
Frank J
FJ
Frey
Perry A
PA
Phillips
George N
GN
D000244:Adenosine Diphosphate / Q000378:metabolism; D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D029681:Arabidopsis Proteins; D010710:Phosphates; D000245:Adenosine Diphosphate Glucose; D000244:Adenosine Diphosphate; D009713:Nucleotidyltransferases; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; C496715:ADP-glucose pyrophosphorylase, Arabidopsis; D051599:Glucose-1-Phosphate Adenylyltransferase; C020658:glutamine-synthetase adenylyltransferase; D015032:Zinc