Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties. Export

@article{citeulike:2774665, 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 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., Mart\'{\i}nez, 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.}, address = {Department of Biochemistry and Molecular Biology, University of Bergen, N-5009, Bergen, Norway.}, author = {Solstad, T. and Flatmark, T.}, citeulike-article-id = {2774665}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/11012685}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=11012685}, issn = {0014-2956}, journal = {European journal of biochemistry / FEBS}, keywords = {deamidation, expression, hpah}, month = {October}, number = {20}, pages = {6302--6310}, posted-at = {2008-05-09 08:55:24}, priority = {5}, title = {Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11012685}, volume = {267}, year = {2000} }

TY - JOUR ID - citeulike:2774665 L3 - citeulike-article-id:2774665 N2 - 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. IS - 20 JF - European journal of biochemistry / FEBS SN - 0014-2956 AD - Department of Biochemistry and Molecular Biology, University of Bergen, N-5009, Bergen, Norway. EP - 6310 TI - Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties. VL - 267 SP - 6302 KW - deamidation KW - expression KW - hpah AU - Solstad, T AU - Flatmark, T PY - 2000/10// UR - http://view.ncbi.nlm.nih.gov/pubmed/11012685 ER -