The enzymic synthesis of the 1-phosphonomethyl isostere of fructose 1,6-diphosphate in which the 1-phosphate (-OPO(3)H(2)) is replaced by the phosphonomethyl group (-CH(2)PO(3)H(2)) is described. The kinetic properties of this fructose diphosphate isostere and of 4-hydroxy-3-oxobutylphosphonic acid, an isostere of dihydroxyacetone phosphate, with aldolase (EC 4.1.2.13), fructose diphosphatase (EC 3.1.3.11) and glycerol phosphate dehydrogenase (EC 1.1.1.8) are described (see Table 1).