The stereochemical course of rabbit liver fructose bisphosphatase (EC 22.214.171.124) was determined by hydrolyzing the substrate analogue (SP)-[l-18O]fructose 1-phosphorothioate 6-phosphate in H217O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATPβS), and analyzing the isotopic distribution of 18O in ATPβS by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.
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