HUMAN PTERIN-4-ALPHA-CARBINOLAMINE DEHYDRATASE DIMERIZATION COFACTOR OF HEPATOCYTE NUCLEAR FACTOR-1-ALPHA - CHARACTERIZATION AND KINETIC-ANALYSIS OF WILD-TYPE AND MUTANT ENZYMES/

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocy te nuclear factor-1 alpha is a protein with two different functions. W e 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., Ka ufman, S., Milstien, S., Naylor, E. W., Greene, C. L. and 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 mon o-, di-, tri-, and tetrameric forms, irrespective of the presence of t he single Cys81. Sulfhydryl-modifying reagents did not affect the acti vity, but rather showed that Cys81 is exposed. Various pterins bind an d quench the tryptophan fluorescence suggesting the presence of a spec ific binding site. The fluorescence is destroyed upon light irradiatio n. Wild-type and the Cys81Ser protein enhance the rate of the phenylal anine hydroxylase assay approximate to 10-fold, a value similar to tha t of native dehydratase from rat liver; the Cys81Arg mutant, in contra st, has significantly lower activity. This is compatible with the hypo thesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spo ntaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihyd ropterin-4a-carbinolamine approximate to 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved thre e-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier , G. and Suck, D. (1995) EMBO J. 14, 2032-2042].