scholarly journals 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

1996 ◽  
Vol 313 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Anne P. DØSKELAND ◽  
Aurora MARTINEZ ◽  
Per M. KNAPPSKOG ◽  
Torgeir FLATMARK
Keyword(s):  
Fusion Protein ◽  
Phenylalanine Hydroxylase ◽  
Specific Activity ◽  
Factor Xa ◽  
Full Length ◽  
Target Sequence ◽  
Enzyme Form ◽  
Linker Peptide ◽  
Substrate Activation ◽  
Human Phenylalanine Hydroxylase

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. Preincubation 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.

scholarly journals 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

1995 ◽  
Vol 306 (2) ◽  
pp. 589-597 ◽  
Author(s):  
A Martinez ◽  
P M Knappskog ◽  
S Olafsdottir ◽  
A P Døskeland ◽  
H G Eiken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Host Cell ◽  
Phenylalanine Hydroxylase ◽  
Specific Activity ◽  
Factor Xa ◽  
Recombinant Enzyme ◽  
Size Exclusion ◽  
Isolation And Characterization ◽  
Human Phenylalanine Hydroxylase

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.

scholarly journals Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation

FEBS Open Bio ◽  
2017 ◽  
Vol 7 (7) ◽  
pp. 1026-1036 ◽  
Author(s):  
João Leandro ◽  
Anne J. Stokka ◽  
Knut Teigen ◽  
Ole A. Andersen ◽  
Torgeir Flatmark
Keyword(s):  
Active Site ◽  
Phenylalanine Hydroxylase ◽  
Substrate Activation ◽  
Human Phenylalanine Hydroxylase

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

Biochemistry ◽  
1985 ◽  
Vol 24 (3) ◽  
pp. 556-561 ◽  
Author(s):  
Simon C. M. Kwok ◽  
Fred D. Ledley ◽  
Anthony G. DiLella ◽  
Kathryn J. H. Robson ◽  
Savio L. C. Woo
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Phenylalanine Hydroxylase ◽  
Full Length ◽  
Complementary Dna ◽  
Human Phenylalanine Hydroxylase

scholarly journals Author Correction: Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Catarina S. Tomé ◽  
Raquel R. Lopes ◽  
Pedro M. F. Sousa ◽  
Mariana P. Amaro ◽  
João Leandro ◽  
...  
Keyword(s):  
Small Angle ◽  
Phenylalanine Hydroxylase ◽  
Conformational Stability ◽  
Full Length ◽  
Wild Type ◽  
X Ray ◽  
X Ray Scattering ◽  
Human Phenylalanine Hydroxylase ◽  
Ray Scattering

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

scholarly journals Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase

2021 ◽  
Vol 22 (12) ◽  
pp. 6539
Author(s):  
María Conde-Giménez ◽  
Javier Sancho
Keyword(s):  
High Throughput Screening ◽  
Phenylalanine Hydroxylase ◽  
Fluorescence Emission ◽  
Full Length ◽  
Metabolic Enzyme ◽  
Chemical Denaturation ◽  
Apparent Lack ◽  
Catalytic Domains ◽  
Human Phenylalanine Hydroxylase ◽  
Equilibrium Unfolding

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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