title: Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.
abstract: Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha 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 approximately 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 approximately 50-70-fold at 4 degrees 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].
authors.affiliation: Department of Biology, University of Konstanz, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Köster
authors.forename: B
authors.initials: B
authors.lastname: Thöny
authors.forename: P
authors.initials: P
authors.lastname: Macheroux
authors.forename: H C
authors.initials: HC
authors.lastname: Curtius
authors.forename: C W
authors.initials: CW
authors.lastname: Heizmann
authors.forename: W
authors.initials: W
authors.lastname: Pfleiderer
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
mesh_terms: D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000378:metabolism; D011485:Protein Binding; D011622:Pterins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D011622:Pterins; D011994:Recombinant Proteins; D014157:Transcription Factors; D001708:Biopterins; D010649:Phenylalanine; C017226:7,8-dihydrobiopterin; D010651:Phenylalanine Hydroxylase; D006836:Hydro-Lyases; C076392:pterin-4a-carbinolamine dehydratase; D003545:Cysteine