Human Pterin‐4α‐Carbinolamine Dehydratase/Dimerization Cofactor of Hepatocyte Nuclear Factor‐1α

Pterin‐4a‐carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor‐1α is a protein with two different functions. We have overexpressed and purified the human wild‐type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53 , 768–774]. The dehydratase behaves as a tetramer on gel filtration, while cross‐linking experiments showed mono‐, di‐, tri‐, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl‐modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild‐type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay ≈ 10‐fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate‐limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6‐dimethyl‐7,8‐dihydropterin‐4a‐carbinolamine ≈50–70‐fold at 4°C and pH 8.5. The results are discussed in view of the recently solved three‐dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14 , 2032–2042].