Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli.
Human phenylalanine hydroxylase (hPAH) contains three tryptophan residues (W120, W187, and W326). All three tryptophan residues were mutated to phenylalanine either as single mutants or in combination, and one tryptophan was also mutated to isoleucine. The mutant enzymes were expressed in Escherichia coli and purified as fusion proteins with maltose-binding protein and a linker region containing a recognition site for the serine protease factor Xa. After cleavage by factor Xa, all mutants were purified to homogeneity, and the kinetic and spectroscopic properties of the proteins were studied. All the proteins had high catalytic activities, but the affinity for phenylalanine was increased for the W1201 and W120F mutants, and decreased for the W187F and W326F mutants. Using steady-state fluorescence spectroscopy, the contributions of the individual tryptophan residues to the total intrinsic fluorescence of the protein were estimated. On the basis of measurements of mutants containing only one tryptophan, it was calculated that W120, W187, and W326 account for approximately 61, 13, and 26% of the total tryptophan fluorescence of hPAH, respectively, while the positions of the emission maxima (335.5-336.5 nm) and the widths at half-height (55-60 nm) of the emission spectra of the individual tryptophans were rather similar. After incubation with phenylalanine, the quantum yield of wild-type hPAH increases by 15%, and the emission maximum is shifted from 336.5 to 347 nm. This effect is mainly due to changes in the W120 emission. On the basis of fluorescence quenching studies, this amino acid is the most surface-exposed of the tryptophan residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Knappskog
P M
PM
Department of Medical Genetics, University of Bergen, Haukeland Hospital, Norway.
Haavik
J
J
D020106:Acrylamide; D000178:Acrylamides; D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D020106:Acrylamide; D001708:Biopterins; D010649:Phenylalanine; D014364:Tryptophan; D010651:Phenylalanine Hydroxylase; C003402:sapropterin