Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase

Abstract

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][6303][6304][6305][6306][6307][6308][6309][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 1 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°C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from t R 34 min to 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the t R 34 min species decreased with a biphasic time-course with t 0.5 -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 1.5 days in 150 mM Tris/HCl, pH 7.0 at 37°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 b-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 (Asn32fiAsp) 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.

Deamidations in Recombinant Human Phenylalanine Hydroxylase

“…Reversed-phase Chromatography-The mutant tetrameric enzymes were 32 P-labeled by phosphorylation with PKA (see below), proteolyzed by trypsin, and incubated at 37°C up to 32 h to determine the rate of deamidation of labile Asn residues in the phosphopeptide(s) (1). At different time points the incubated peptide mixture was subjected to reversed-phase chromatography using a ConstaMetric gradient System (Laboratory Data Control) and a 4.6-mm ϫ 10-cm Hypersil ODS C18 column (Hewlett-Packard) fitted with a 2-cm guard column of the same material.…”

“…Solvent A was 50 mM ammonium acetate (pH 8.0) and solvent B 50 mM ammonium acetate in 70% (v/v) acetonitrile (pH 8.0). A linear gradient of 10 -50% solvent B was used at a flow rate of 1 ml/min for 60 min (1). Samples were collected every 15 s, and the elution pattern of phosphopeptides was analyzed by liquid scintillation counting and resolved into individual components, using the Peak Fit software program (SPSS Inc., Chicago); the "auto-fit II-Residuals" was used with the confidence level set at Ն95%.…”

“…Upon its phosphorylation with PKA and tryptic proteolysis, the 32 Plabeled peptides were resolved by reversed-phase chromatography into some minor phosphopeptides and a major component (t R ϳ34 min), which revealed a time-dependent shift to a more hydrophilic position (t R ϳ31 min) when incubated at 37°C (1). When the phosphopeptides resulting from tryptic proteolysis of the N32D and G33A/G33V mutant enzymes were subjected to reversed-phase chromatography, the major peptide of N32D revealed the expected retention time of t R ϳ31 min, while that of G33A/G33V mutant derived peptide eluted with a t R ϳ36 min, and none of them shifted on further incubation at pH 7 and 37°C (Fig.…”

“…Thus, the microheterogeneity pattern is highly dependent on the induction time with IPTG in E. coli (4). Due to the relatively high rate of deamidation, the labile amide-containing residues have been considered to be Asn residues (4), and this conclusion has recently been confirmed by the demonstration of iso-Asp in several tryptic peptides of the highly deamidated full-length wt-hPAH (1). In the deamidation reaction Asn is converted to Asp and iso-Asp in a variable ratio, sometimes with iso-Asp as the main product (5,6).…”

“…Isoelectric focusing and two-dimensional electrophoresis of recombinant hPAH expressed in E. coli for 24 h at 28°C revealed five components of decreasing staining intensity and decreasing pI (denoted hPAH I-V). This microheterogeneity was shown to be the result of nonenzymatic deamidations of Asn residues (1,4). Mainly one band with the highest pI (hPAH I) was, however, detected after a short induction period of 2 h at 28°C, and this form was considered to represent the newly synthesized and most native, nondeamidated form of the enzyme.…”