The combmed effect of tautomerlzatlon and enzymatic converslon on the phosphorylatlon of o-fructose by hexokmase has been studled A computer model has been developed to describe the simultaneous enzymatic and tautomerlzatlon reactions m small lmmoblhzed enzyme reactors The tautomer composition of fructose was calculated from available rate constants, takmg mto account cr-fructofuranose, p-fructofuranose, B-fructopyranose, and the open-chain keto form The conversion to fructose-6-phosphate predlcted by the model was compared with conversions determined expenmentally m a flow-mlectlon system An lteratlon procedure permltted the determmatlon of the enzymatic rate constant as well as the rate constants for ring openmg and nng closing of the p-fructopyranose anomer, which were 0 038 and 4 29 s-l, respectively Each tautomer and tautomer combination was tested as possible candidates for the enzymatic phosphoIylation The only alternatives consistent with experlmental data were reaction with &fructofuranose alone or m combmatlon with the a-form From structural arguments the p-form 1s more hkely to be the substrate, as shown m the literature The slow rmg-opening of B-fructopyranose complicates fructose determmatlons using rapid, tautomer-selective enzymatic methods