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 bra in catalyses the hydrolysis of the phosphate ester of a range of purin e- and pyrimidine-containing nucleoside 2'-phosphates including adenos ine 2'-phosphate (2'-AMP) but not adenosine 2'-phosphorothioate (2'-AM PS). 2'-AMPS also fails to serve as an inhibitor under these condition s. In contrast to the situation for the alcohol hydrolysis product, in ositol, adenosine does not serve as a product inhibitor for the enzyme or mediate the enzyme-catalysed exchange of O-18-label from [O-18]wat er into inorganic phosphate. However, in the presence of Mn2+ ions 2'- AMPS is a substrate for the enzyme. These findings indicate that the p roduct adenosine does not bind to the enzyme in its ground-state confo rmations and that a strong phosphate group-holoenzyme interaction is r equired to stabilise a high-energy arrangement in the enzyme-substrate complexes of 2'-AMPS and, probably, 2'-AMP. On the basis of these res ults and those from previous kinetic and substrate modification studie s 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 rib ofuranosyl position through the direct chelation of the second Mg2+ io n to the bridging phosphate ester 2'-O-atom and the ribofuranose ring O-atom. An alternative high-energy arrangement in which the interactio n of the second Mg2+ ion with the ribofuranose ring O-atom is mediated via water, such that the conformational strain in the furanose ring i s relaxed, but where the entropy of the water is decreased, is also a possibility.