Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
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PMID:Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli. 754 12

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.
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PMID:Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. 788 15

The phosphorylation of human phenylalanine hydroxylase by cyclic AMP-dependent protein kinase was studied using recombinant enzyme expressed as a fusion protein in the pMAL system of Escherichia coli. Using the target sequence of the restriction protease enterokinase (Asp4-Lys) as the linker peptide, 100% full-length human phenylalanine hydroxylase was obtained on protease cleavage. The fusion protein and human phenylalanine hydroxylase were both phosphorylated at Ser-16 with a stoichiometry of 1 mol of Pi/mol of subunit. The rate of phosphorylation of human phenylalanine hydroxylase was inhibited about 40% by the cofactor tetrahydrobiopterin, and this inhibition was completely prevented by the simultaneous presence of L-phenylalanine (i.e. at turnover conditions). Phosphorylated enzyme revealed a 1.6-fold higher specific activity than the non-phosphorylated enzyme form, and it also required a lower concentration of L-Phe for substrate activation. Pre-incubation with L-Phe increased the specific activity of phenylalanine hydroxylase 2- to 4-fold, L-Phe acting with positive cooperativity. Thus, the basic catalytic and regulatory properties of recombinant human phenylalanine hydroxylase, as well as those observed for the enzyme as a fusion protein, are similar to those previously reported for the rat liver enzyme. When the target sequence of the restriction protease factor Xa (Ile-Glu-Gly-Arg) was used as the linker between maltose-binding protein and human phenylalanine hydroxylase, cleavage of the fusion protein gave a mixture of full-length hydroxylase and a truncated form of the enzyme lacking the 13 N-terminal residues. Interestingly, phosphorylation of the fusion protein, before exposure to factor Xa, almost completely protected against secondary cleavage by this restriction protease at Arg-13 of phenylalanine hydroxylase.
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PMID:Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease. 857 72

The G46S mutation in the phenylalanine hydroxylase (PAH) gene was identified by fluorescence-based single-strand conformation polymorphism (F-SSCP) analysis on phenylketonuria (PKU) haplotype 5.9 alleles. DNA sequencing of PAH exon 2 revealed a G-to-A transition in cDNA position 136. G46S mutations were present on 17 of 236 Norwegian PKU alleles (7.2%) and on 8 of 176 Swedish PKU alleles (4.5%). Analysis of all 13 exons with the flanking regions further detected a 1316-35c > t polymorphism (PAH intron 12), associated with both G46S and haplotype 5.9. Three patients were homozygous for the G46S mutation, two were untreated and had mild and severe mental retardation, respectively. The G46S mutation was introduced in the PAH cDNA by site-directed mutagenesis and expressed in three different systems (the pMAL/Escherichia coli system, the pcDNA3/human embryonic kidney (A293) cells, and the pcDNA3/TnT coupled in vitro transcription-translation system). The mutant recombinant E. coli fusion protein was recovered in high yield and with a specific activity of the purified tetrameric form, which was higher than the wild-type activity. After transient expression in A293 cells, the amount of the G46S protein was only about 3% of the wild type at equal PAH mRNA levels. The fusion protein cleaved by restriction protease factor Xa, as well as the enzyme produced by in vitro transcription-translation, revealed an abnormal susceptibility to form catalytically inactive high-molecular-mass aggregates of the enzyme. This aggregation, followed by an increased cellular degradation of the G46S mutant enzyme, is compatible with the clinical/metabolic phenotype of the affected homozygous and compound heterozygous patients.
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PMID:PKU mutation G46S is associated with increased aggregation and degradation of the phenylalanine hydroxylase enzyme. 882 56