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

The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH ) was investigated by isoelectric focusing and 2D electrophoresis. When exp ressed in Escherichia coli four main components (denoted hPAH I-IV) of appr oximate to 50 kDa were observed on long-term induction at 28-37 degrees C w ith 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-translatio n system, including both its nonphosphorylated and phosphorylated forms whi ch were separated on the basis of a difference in mobility on SDS/PAGE. Exp erimental evidence is presented that the microheterogeneity is the result o f nonenzymatic deamidations of labile amide containing amino acids. When ex pressed in E. coli at 28 degrees C, the percentage of the acidic forms of t he 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 conversio n to the acidic components (hPAH II-IV) revealed a pH and temperature depen dence characteristic of a nonenzymatic deamidation of asparagine residues i n proteins, with the release of ammonia. Comparing the microheterogeneity o f 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 dom ain as defined by crystal structure analysis [Erlandsen, H., Fusetti, F., M artinez, A., Hough, E., Flatmark, T. & Stevens, R. C. (1997) Nat. Struct. B iol. 4, 995-1000]. It is further demonstrated that the progressive deamidat ions which occur in E. coli results in a threefold increase in the catalyti c efficiency (V-max/[S](0.5)) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzym e. The results also suggest that deamidation may play a role in the long te rm regulation of the catalytic activity and the cellular turnover of this e nzyme.