An inhibitory regulatory domain in phenylalanine hydroxylase (PAH), located in a position roughly between 1 and 100 amino acid residues, inhibits PAH activity more than 10-fold when the enzyme is assayed with the naturally-occurring coenzyme, tetrahydrobiopterin (BH4). The inhibitory effect disappears when 6MPH4, an analog of BH4, is used as the coenzyme. In order to define the region in PAH which plays a key role in the inhibition, deletion mutations were generated. We generated three mutants, N-15, N-26 and N-50. Compared to wild-type, Vmax for N- 15 increased 2-fold, while for N-26, it increased 12-fold. Further deletion, N-50, does not show significant differences compared to N-26 in Vmax but much less in Km with BH4 (1.8-fold). These results show that the region between position 15 and 26 plays an important role in the negative regulation of PAH. Right now this region is being further studied by site directed mutagenesis. It was previously shown that experimental diabetes in rats leads to increased levels of PAH. We have now shown in collaboration with Dr. John Donlon in Ireland, that this condition leads to an increase in hepatic levels of BH4. Since diabetes is known to increase gluconeogenesis, we examined the effect of feeding rats gluconeogenic precursors (e.g., glycerol or fructose). These regimens led to a decrease in the hepatic levels of both PAH and BH4, an indication that the hydroxylase and its pterin cofactor are coordinately regulated in vivo. - phenylalanine hydroxylase, tetrahydrobiopterin, BH4, diabetes, gluconeogenesis, glycerol, fructose