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T Flatmark

Publications and source records attributed to T Flatmark.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection.

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

Animals↗
PubMed1998

Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol inhibitors at 2.0 A resolution.

The aromatic amino acid hydroxylases represent a superfamily of structurally and functionally closely related enzymes, one of those functions being reversible inhibition by catechol derivatives. Here we present the crystal structure of the dimeric catalytic domain (residues 117-424) of human phenylalanine hydroxylase (hPheOH), cocrystallized with various potent and well-known catechol inhibitors and refined at a resolution of 2.0 A. The catechols bind by bidentate coordination to each iron in both subunits of the dimer through the catechol hydroxyl groups, forming a blue-green colored ligand-to-metal charge-transfer complex. In addition, Glu330 and Tyr325 are identified as determinant residues in the recognition of the inhibitors. In particular, the interaction with Glu330 conforms to the structural explanation for the pH dependence of catecholamine binding to PheOH, with a pKa value of 5.1 (20 degreesC). The overall structure of the catechol-bound enzyme is very similar to that of the uncomplexed enzyme (rms difference of 0.2 A for the Calpha atoms). Most striking is the replacement of two iron-bound water molecules with catechol hydroxyl groups. This change is consistent with a change in the ligand field symmetry of the high-spin (S = 5/2) Fe(III) from a rhombic to a nearly axial ligand field symmetry as seen upon noradrenaline binding using EPR spectroscopy [Martinez, A., Andersson, K. K., Haavik, J., and Flatmark, T. (1991) Eur. J. Biochem. 198, 675-682]. Crystallographic comparison with the structurally related rat tyrosine hydroxylase binary complex with the oxidized cofactor 7,8-dihydrobiopterin revealed overlapping binding sites for the catechols and the cofactor, compatible with a competitive type of inhibition of the catechols versus BH4. The comparison demonstrates some structural differences at the active site as the potential basis for the different substrate specificity of the two enzymes.

Binding, Competitive↗
PubMed1998

Partial characterization and three-dimensional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria.

The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.

Amino Acid Sequence↗
PubMed1998

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria.

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.

Amino Acid Sequence↗
PubMed1998

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

Binding Sites↗
PubMed1997

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase.

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.

Crystallization↗
PubMed1997

Dopamine oxidation generates an oxidative stress mediated by dopamine semiquinone and unrelated to reactive oxygen species.

Dopamine (100 microM, 10-30 min) inhibits/inactivates the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The dopamine dependent inhibition was enhanced by adding soluble dopamine beta-monooxygenase (DBM, 0.2 U/ml) and completely prevented by ascorbate (1 mM), dithiothreitol (2 mM) and approximately 80% by the DBM inhibitor fusaric acid (10 microM). This indicates that the inhibition is caused by the dopamine semiquinone free radical generated during DBM-dependent dopamine oxidation. Catalase, superoxide dismutase or both did not prevent the inhibition, and DBM-catalysed dopamine oxidation did not change the basal level of lipid peroxidation, excluding the involvement of reactive oxygen species as being responsible for the inhibition. N-ethylmaleimide-sensitive ATPase activity (i.e. the proton translocating ATPase) in the vesicle membranes was inhibited during dopamine incubation, indicating that the toxic metabolite (dopamine semiquinone) inhibits proton pumping by inhibiting the endogenous vacuolar H(+)-ATPase. As this proton pump represents the driving force for the vesicular uptake and storage of catecholamines, the dopamine dependent inhibition, if taking place in vivo, may inhibit dopamine uptake in storage vesicles in sympathetic neurons, e.g. as observed in the myopathic hamster heart.

Animals↗
PubMed1997

Generation of reactive oxygen species by tyrosine hydroxylase: a possible contribution to the degeneration of dopaminergic neurons?

It has been suggested that idiopathic parkinsonism, characterized by a loss of dopaminergic neurons of the nigrostriatal pathway, is due to the intracellular generation of reactive oxygen species, generated by a nonenzymatic or enzymatic partial reduction of dioxygen. Based on in vitro studies of the iron-containing monooxygenase tyrosine hydroxylase (TH), evidence is presented that this enzyme system may also contribute to such an oxidative stress. Thus, the purified and Fe(2+)-reconstituted recombinant human enzyme shows a time- and temperature-dependent partial uncoupling of the hydroxylation of L-tyrosine with the natural cofactor (6R)-tetrahydrobiopterin, resulting in the formation of H2O2. The degree of uncoupling of the hydroxylation reaction is significantly higher when certain substrate analogues, notably the 7-substituted isomer (7-tetrahydrobiopterin) of the natural cofactor, is used. In the presence of H2O2 and Fe2+, the addition of TH increases the production of the highly reactive.OH radical, probably via a Fenton type of reaction. It is not clear whether this in vitro reaction can mediate cellular injury in vivo. However, it is known that the distribution of TH in the central and peripheral nervous system often corresponds to that of the neuronal degeneration in idiopathic parkinsonism, a finding that is compatible with a pathogenetic effect of TH.

Dopamine↗
PubMed1997

Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion.

Purification of herpes simplex virus type 1 (HSV-1) is often performed by centrifugation in gradients of various materials. A major problem with such procedures is a gradual decrease in the infectivity of the virus, probably due to the influence of the gradient material. In the present work we have compared Nycodenz gradients with Ficoll gradients for HSV-1 purification. Both gradient materials have low osmolarities. The purity of the preparations recovered from the two gradients was similar, as measured by electron microscopy and two-dimensional gel electrophoresis, and the yield of infectious virus was approximately the same. Szilágyi & Cunningham (22) reported previously that Ficoll gradients separate one band of complete virions and another band containing light particles devoid of nucleocapsids. In Nycodenz gradients we observed the same bands, and an additional third band which contained aggregates and disrupted particles. In this respect, the separation in Nycodenz is slightly superior to that in Ficoll. The purified HSV-1 virions were subjected to analytical differential centrifugation and an S-value of 670S was calculated for the virus.

Animals↗
PubMed1997

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes.

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme.

Amino-terminal and carboxy-terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452). The wild-type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L-Phe) binding (Hill coefficient h = 2.2, S0.5 = 154 microM). The deletion mutants lacking the carboxy-terminal 24 amino acids hPAH (Ser2-Gln428) and hPAH(Gly103-Gln428) formed catalytically active dimers, and incubation with L-Phe did not promote the formation of tetramers, a characteristic property of dimeric wt-hPAH. The carboxyterminus thus seems to contain a motif required for dimer-dimer interaction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Ser2-Gln428) and hPAH(Gly103-Gln428) were all activated by prior incubation with L-Phe, but did not reveal any positive cooperativity of substrate binding (h = 1.0). The activation by L-Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino-terminal sequence. i.e. wt-hPAH and the Ser2-Gln428 mutant. The amino-terminal deletion mutants hPAH(Asp112-Lys452) and hPAH(Gly103-Gln428) revealed high specific activity, increased apparent affinity for L-Phe (S0.5 = 60 microM) and a tryptophan fluorescence emission spectrum similar to that of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt-hPAH amino-terminal sequence. Our results are compatible with a model in which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino-terminal domain to the high-affinity binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.

Allosteric Regulation↗
PubMed1996

Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system.

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7-8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.

Animals↗
PubMed1996

Characterization of a novel pterin intermediate formed in the catalytic cycle of tyrosine hydroxylase.

A novel pterin intermediate, in addition to the expected 4a-hydroxytetrahydrobiopterin (4a-OH-BH4) and quinonoid dihydrobiopterin, was generated during catalytic turnover of tyrosine hydroxylase (TH) with tetrahydrobiopterin as the cofactor. Based on chromatographic, spectroscopic and stability properties its structure is proposed to be similar to the product formed by the non-enzymic conversion of synthetic 4a-OH-BH4 [Bailey, Rebrin, Boerth and Ayling (1995) J. Am. Chem. Soc. 117, 10203-10211]. This compound was tentatively described as a 4a-adduct of a side-chain hydroxy group, i.e. the O2', 4a-cyclic-tetrahydrobiopterin (4a-Cyc-BH4). The intermediate generated in the TH reaction has a UV spectrum which is similar to that of 4a-OH-BH4, but elutes with a longer retention time (tR = 1.69 min compared with 1.06 min) on reversed-phase chromatography. Its conversion into quinonoid dihydrobiopterin is catalysed by pterin-4a-carbinolamine dehydratase (EC 4.2.1.96), although 4a-OH-BH4 is the preferred substrate for that enzyme. A precursor-product relationship was demonstrated between 4a-OH-BH4 and the putative 4a-Cyc-BH4 intermediate. The apparent stability of this compound is dependent on pH as well as on the nature of the buffer ions. At pH 8.0 a large amount was generated in Hepes and Tris, but little in phosphate buffer. At pH 7.0 in Hepes (standard assay conditions) and Tris buffer the putative 4a-Cyc-BH4, but no 4a-OH-BH4, was observed. None of the intermediates was observed at pH 6.0. The accumulation of these intermediates in the absence of dehydratase has important implications for the assay of TH and phenylalanine hydroxylase activities, and is also compatible with a possible physiological role of the dehydratase in the synthesis of catecholamines in vivo.

Biopterins↗
PubMed1996

Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation.

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.

Catecholamines↗
PubMed1996

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.

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.

Amino Acid Sequence↗
PubMed1996

PKU mutation G46S is associated with increased aggregation and degradation of the phenylalanine hydroxylase enzyme.

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.

Adult↗