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Article

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

Jason G. McCoy,^§ Abolfazl Arabshahi,^§ Eduard Bitto, Craig A. Bingman, Frank J. Ruzicka,^§ Perry A. Frey,^§ and George N. Phillips, Jr.*^§

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, and University of Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, Wisconsin 53706

Biochemistry, 2006, 45 (10), pp 3154–3162

DOI: 10.1021/bi052232m

Publication Date (Web): February 17, 2006

†

Supported by NIH Grant GM30480 (P.A.F.), NIH Protein Structure Initiative Grants GM074901 and GM64598 (John L. Markley, principal investigator), and NLM Training Grant LM007359 (J.G.M.). Use of the Advanced Photon Source and the Argonne National Laboratory Structural Biology Center and sector-32 beamlines was supported by the U.S. Department of Energy, Office of Energy Research, under Contract W-31-109-ENG-38. Use of BioCARS was supported by the National Institutes of Health, National Center for Research Resources, via Grant RR07707. We gratefully acknowledge the Michigan Economic Development Corp. and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817).

‡

X-ray coordinates for the Arabidopsis At5g18200.1 protein have been deposited in the Protein Data Bank as entries 1ZWJ and 1Z84.

Department of Biochemistry.

Center for Eukaryotic Structural Genomics.

To whom correspondence should be addressed: Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Telephone: (608) 263-6142. E-mail: Phillips@ biochem.wisc.edu.

Abstract

The X-ray crystal structure of the At5g18200.1 protein has been determined 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 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 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 a k_cat of 4.1 s^-¹ and K_m values of 1.4 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 a k_cat of 2.7 s^-¹ and K_m values of 6.9 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 determined 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.