The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO(2)) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-(13)C]glucose, [U-(13)C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive ( K(i)=180 microM) and uncompetitive ( K'(i)=350 microM) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively ( K(i)=1.3 microM) with a low contribution of uncompetitive inhibition ( K'(i)=13 microM). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 microM) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 microM). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.