Authors: Solstad, Therese; Flatmark, Torgeir

Source: FEBS Journal, Volume 267, Number 20, October 2000 , pp. 6302-6310(9)

Publisher: Wiley-Blackwell

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 °C with isopropyl thio-β-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 °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 °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 (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.

Keywords: phenylalanine hydroxylase; heterologous expression; microheterogeneity; deamidation; Escherichia coli.

Document Type: Research article

DOI: http://dx.doi.org/10.1046/j.1432-1327.2000.01715.x

Affiliations: 1: Department of Biochemistry and Molecular Biology, University of Bergen, N-5009, Bergen, Norway

Publication date: 2000-10-01

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• In this Subject: Anatomy & Physiology ,  Biology ,  Chemistry (General) ,  Biochemistry
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