The protein phosphatase inhibiting toxins microcystin and nodularin act rapidly to induce apoptotic cell death. Their inhibitory effect on protein phosphatases 1 and 2A can be utilized as tools to understand the phosphorylation-dependent regulatory mechanism underlying the early stage of apoptosis. The incubation of freshly isolated hepatocytes with these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appears [Fladmark, K. E., Brustugun, O. T., Hovland, R., Boe, R., Gjertsen, B. T., Zhivotovsky, B. and Doskeland, S. O. (1999) Cell Death Differ. 6, 1099-108]. Proteins subjected to phosphorylation in this early phase of apoptosis may play key roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-induction by microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainly serine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins reduces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectrometric identification and characterization. We will here give an overview of our strategy for identification of low-abundance phosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 °C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn32 in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly33(Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 °C) expressed enzyme has shown Asn376 and Asn133 to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn32 and Asn376 are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn32 → Asp, Asn376 → Asp, and the double mutant forms expressed for 2 h at 28 °C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn32 in the wt-hPAH (24 h expression at 28 °C) and the Asn32 → Asp mutation both increase the initial rate of phosphorylation of Ser16 by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly33 with Ala or Val, both preventing the deamidation of Asn32, resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn → Asp substitutions (in the catalytic domain) revealed that Asn207 and Asn223 have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied,i.e. Asn167 → Ile and Asn207 → Asp, and their phenotypes were characterized.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.

Phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, EC 1.14.16.1) catalyzes a single hydroxylation by an intermolecular oxygenation reaction of an aromatic ring (L-phenylalanine, L-Phe) in an Fe(II)- and pterin-dependent fashion (reviewed in (1,2)). X-ray crystal structure analyses of human and rat PAH (hPAH and rPAH) have revealed that the monomer is organized into three main domains, i.e. a N-terminal regulatory domain, a catalytic domain and a small C-terminal dimerization/tetramerization domain (reviewed in (2)). PAH isolated from rat liver and as recombinant human enzyme are both in a tetramer dimer equilibrium (3,4). The tetrameric form is a dimer of dimers with defined interphases between the subunits in the dimers. The 41 C-terminal residues that comprise the tetramerization motif are organized into an “arm” consisting of two short ß strands, forming a ß ribbon, and a 40-Å long α helix (Gln428-Lys452)(5). The helices interact with each other, forming an anti-parallel coiled coil structure in the center of the tetramers and mutually switch their position to promote tetramerization (5). A comparison of the crystal structure of hPAH (5,6) and rPAH (7) have revealed the same overall fold for the highly conserved catalytic domain of the two enzyme forms. Thus, the human and rat enzyme share 96

The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH) was investigated by isoelectric focusing and 2D electrophoresis. When expressed in Escherichia coli four main components (denoted hPAH I-IV) of approximately 50 kDa were observed on long-term induction at 28-37 degrees C with isopropyl thio-beta-D-galactoside (IPTG), differing in pI by about 0.1 pH unit. A similar type of microheterogeneity was observed when the enzyme was expressed (1 h at 37 degrees C) in an in vitro transcription-translation system, including both its nonphosphorylated and phosphorylated forms which were separated on the basis of a difference in mobility on SDS/PAGE. Experimental evidence is presented that the microheterogeneity is the result of nonenzymatic deamidations of labile amide containing amino acids. When expressed in E. coli at 28 degrees C, the percentage of the acidic forms of the enzyme subunit increased as a function of the induction time with IPTG, representing about 50% on 8 h induction. When the enzyme obtained after 2 h induction (containing mainly hPAH I) was incubated in vitro, its conversion to the acidic components (hPAH II-IV) revealed a pH and temperature dependence characteristic of a nonenzymatic deamidation of asparagine residues in proteins, with the release of ammonia. Comparing the microheterogeneity of the wild-type and a truncated form of the enzyme expressed in E. coli, it is concluded that the labile amide groups are located in the catalytic domain as defined by crystal structure analysis [Erlandsen, H., Fusetti, F., Martínez, A., Hough, E., Flatmark, T. & Stevens, R. C. (1997) Nat. Struct. Biol. 4, 995-1000]. It is further demonstrated that the progressive deamidations which occur in E. coli results in a threefold increase in the catalytic efficiency (Vmax/[S]0.5) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzyme. The results also suggest that deamidation may play a role in the long term regulation of the catalytic activity and the cellular turnover of this enzyme.