Studies on the Divalent Cation Binding Site in ADP-glucose Pyrophosphorylase

A.A. IGLESIAS; M.C. ESPER; M. ALEANZI; M.A. BALLICORA; J. PREISS

Iguazú, Misiones, Argentina

Congreso; XL Reunión de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2004

SAIB

BIOCELL 28 (Suppl.), 2004 75ES-P15.STUDIES ON THE DIVALENT CATION BINDING SITE IN ADP-GLUCOSE PYROPHOSPHORYLASEIglesias AA, Esper MC, Aleanzi M, Ballicora MA*, Preiss J*. Lab. Enzimología Molecular, Fac. Bioquímica y Cs. Biológicas, UNL, Paraje "El Pozo", S3000ZAA, Santa Fé, Argentina; and *Dept. Biochemistry & Mol. Biol., Michigan State University., E. Lansing, MI 48824, USA. E-mail: iglesias@fbcb.unl.edu.arADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key regulatory step in the synthesis of glycogen and starch in bacteria and plants, respectively. The enzyme requires a divalent cation (physiologically Mg2+) as an essential cofactor. Several studies have been performed to identify amino acid residues and structural domains in the protein involved in the binding of subtrates (Glc1P and ATP) and allosteric effectors, as well as in catalysis. The crystal structure of a small subunit of potato tuber ADP-Glc PPase is close to be solved. So far, little information is available concerning residues and structural domains involved in the binding of the divalent cation to the enzyme. We performed affinity cleavage by Cu2+ on the ADP-Glc PPase from Escherichia coli. The cation Cu2+, in the presence of H2O2 and ascorbate, cleaved the enzyme mainly in two sites, which localization was determined by the use of a mutant enzyme and toward mass spectrometry analysis of the resulting peptides. The main cleavage occurred between residues D233 and E234, and a second cleavage (enhanced by ATP) took place around K39. Results agree with the hypothesis that the enzyme may have two metal binding siteslocated in opposite sides in the 3-D model of ADP-Glc PPase.