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The natural cofactor of phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH4), regulates the enzyme activity as well as being essential in catalysis. BH4-responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine. Here, we showed in a coupled transcription–translation in vitro assay that upon expression in the presence of BH4, wild-type PAH enzyme activity was enhanced. We then investigated the effect of BH4 on PAH activity in transgenic mice that had a complete or partial deficiency in the endogenous cofactor biosynthesis. The rate of hepatic PAH enzyme activity increased significantly with BH4 content without affecting gene expression or Pah-mRNA stability. These results indicate that BH4 has a chaperon-like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto-inactivation and degradation also in vivo. Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH4 on an additional level and provide a molecular explanation for cofactor-responsive PKU.

The 14-3-3 proteins are important effectors of Ser/Thr phosphorylation in eukaryotic cells. Using mathematical modelling we investigated the roles of these proteins as effectors in signalling pathways that involve multi-phosphorylation events. We defined optimal conditions for positive and negative cross-talk. Particularly, synergistic signal interaction was evident at very different sets of binding affinities and phosphorylation kinetics. We identified three classes of 14-3-3 targets that all have two binding sites, but displayed synergistic interaction between converging signalling pathways for different ranges of parameter values. Consequently, these protein targets will respond differently to interventions that affect 14-3-3 binding affinities or phosphorylation kinetics. publishedVersion

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% α-helix, 28% extended structures, 12% β-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.

Wild-type recombinant human phenylalanine hydroxylase (wt-hPAH) is activated about 1.5-fold by exposure to alkaline pH (pH 8.5–9.0). In order to study whereas this activation might be related to the activation of the rat enzyme by N-ethylamaleimide-modification of Cys237 [Gibbs and Benkovic (1991) Biochemistry 30, 6795], mutant proteins of hPAH with Cys237 changed to Ser (S) or Glu (D) have been prepared. The mutant forms have high specific activity at pH 7.0 and high affinity for l-Phe, notably for hPAH-C237D, which shows a 3-fold higher activity than l-Phe-activated wt-hPAH and it is not further activated by pre-incubation with l-Phe. Moreover, the emission maximum of the intrinsic fluorescence of hPAH-C237D (λmax em=347 nm) resembles that of activated forms of wt-hPAH. However, the activity of this mutant at neutral pH is further activated by exposure to alkaline pH, indicating that activation of wt-hPAH by alkaline pH is not restricted to ionization of Cys237.

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.

The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure–function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.

The tetrahydrobiopterin-dependent iron-enzyme tyrosine hydroxylase (TH) catalyses the rate-limiting step in catecholamine biosynthesis. Electron paramagnetic resonance (EPR) data show that following phosphorylation of Ser-40 by protein kinase A, the enzyme-bound Fe(III), coordinated to catecholamines, can be reduced by 6-methyl-tetrahydropterin under turnover conditions. The 8-fold increase in product formation upon phosphorylation can partly be explained by an increase in the fraction of active TH, by dissociation of the endogenous catecholamine inhibitors.

Bovine adrenal tyrosine hydroxylase (TH) is isolated in a partially inhibited state with the feed-back inhibitors adrenaline and noradrenaline tightly coordinated to high-spin (S = 5/2) Fe(III) at the active site. In addition to the charge-transfer interaction with iron, an additional charged group in the polypeptide chain, with an apparent pKa of about 5.3 at 4 degrees C, is involved in the binding of catecholamines. Protonation of this group increases the pseudo-first order rate constant for the dissociation of the TH-[3H]noradrenaline complex more than 100-fold at 4 degrees C. At pH 7.0 and 30 degrees C, phosphorylation of Ser-40 causes a 6-fold increase in the rate constant for this dissociation.