Research article

The Metal Coordination of sCD39 during ATP Hydrolysis

Wei Chen and Guido Guidotti

Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA

author email corresponding author email

BMC Biochemistry 2001, 2:9doi:10.1186/1471-2091-2-9

Published:	12 September 2001

Abstract

Background

The hydrolysis of ATP and ADP by ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) requires divalent cations, like Ca²⁺ and Mg²⁺. In spite of considerable work, it is not clear whether divalent cations bind to the enzyme in the absence of nucleotide or only as nucleotide-Me⁺² complex. Here we study the protein ligands for Me⁺².

Results

When VO²⁺ was used as a substitute for Ca²⁺, the ATPase activity of soluble CD39 was 25% of that with Ca²⁺ as cofactor. Protein ligands of the VO²⁺-nucleotide complex bound to the catalytic site of soluble CD39 were characterized by electron paramagnetic resonance (EPR) spectroscopy. The EPR spectrum contained one species designated T with VO²⁺-AMPPNP as ligand. Two species D1 and D2 were observed when VO²⁺-AMPCP was bound to soluble CD39. The results suggest that species D1 and D2 represent the metal-ADP complexes at the catalytic site of soluble CD39 corresponding to the intermediate formed during ATP hydrolysis and the substrate for further hydrolysis, respectively.

Conclusions

VO²⁺ can functionally substitute for Ca²⁺ as a cofactor of sCD39, and it produces four different EPR features when bound in the presence of different nucleotides or in the absence of nucleotide. The metal coordination for each conformation corresponding to each EPR species is proposed, and the mechanism of sCD39 catalysis is discussed.