cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.

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http://dx.doi.org/10.1046/j.1471-4159.1996.67030917.xDOI Listing

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