Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids

Abstract

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 < 50 kDa were observed on long-term induction at 28±37 8C with isopropyl thio-b-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 8C) 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 8C, 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., Martõ Ânez, 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 (V max /[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.

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

“…After 5‐min preincubation with l ‐Phe, the reaction was initiated by adding BH 4 with DTT, and allowed to proceed as described 25. The amount of L‐Tyr formed after one minute (standard), or other selected time points, was measured by HPLC with fluorimetric detection 22.…”

Deamidations in Recombinant Human Phenylalanine Hydroxylase

“…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. 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).…”

“…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.…”

“…Mutations in the human enzyme (hPAH) leading to altered kinetic properties or reduced stability of the enzyme are associated with the autosomal recessive disorder phenylketonuria (PKU). hPAH isolated from liver (2) and as recombinant enzyme expressed in Escherichia coli (3,4) has been found to exist as a mixture of 4 -5 molecular forms with the same apparent subunit molecular mass, but with different isoelectric points (pI). 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).…”

“…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).…”