ACTIVE CONFORMATION OF THE INOSITOL MONOPHOSPHATASE SUBSTRATE, ADENOSINE 2'-PHOSPHATE - ROLE OF THE RIBOFURANOSYL O-ATOMS IN CHELATING A 2ND MG2+ ION

In the presence of Mg2+ ions, inositol monophosphatase from bovine brain catalyses the hydrolysis of the phosphate ester of a range of purine- and pyrimidine-containing nucleoside 2'-phosphates including adenosine 2'-phosphate (2'-AMP) but not adenosine 2'-phosphorothioate (2'-AMPS). 2'-AMPS also fails to serve as an inhibitor under these conditions. In contrast to the situation for the alcohol hydrolysis product, inositol, adenosine does not serve as a product inhibitor for the enzyme or mediate the enzyme-catalysed exchange of O-18-label from [O-18]water into inorganic phosphate. However, in the presence of Mn2+ ions 2'-AMPS is a substrate for the enzyme. These findings indicate that the product adenosine does not bind to the enzyme in its ground-state conformations and that a strong phosphate group-holoenzyme interaction is required to stabilise a high-energy arrangement in the enzyme-substrate complexes of 2'-AMPS and, probably, 2'-AMP. On the basis of these results and those from previous kinetic and substrate modification studies it is proposed that a second Mg2+ ion might stabilise a conformation in which the adenine moiety of bound 2'-AMP occupies a C-1'-axial ribofuranosyl position through the direct chelation of the second Mg2+ ion to the bridging phosphate ester 2'-O-atom and the ribofuranose ring O-atom. An alternative high-energy arrangement in which the interaction of the second Mg2+ ion with the ribofuranose ring O-atom is mediated via water, such that the conformational strain in the furanose ring is relaxed, but where the entropy of the water is decreased, is also a possibility.