EXPRESSION AND DELETION MUTAGENESIS OF TRYPTOPHAN-HYDROXYLASE FUSION PROTEINS - DELINEATION OF THE ENZYME CATALYTIC CORE/

cDNAs encoding the full-length sequence for tryptophan hydroxylase, an d 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 wit hin minutes by affinity chromatography on glutathione-agarose, The ful l-length enzyme and the catalytic core expressed very high levels of t ryptophan hydroxylase activity. The regulatory domain was devoid of ac tivity, The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and th e kinetic properties of each recombinant enzyme for cofactor and subst rate compared very closely to native, brain tryptophan hydroxylase, Bo th active forms of the glutathione S-transferase-tryptophan hydroxylas e fusion proteins had strict requirements for ferrous iron in catalysi s and expressed much higher levels of activity (V-max) than the brain enzyme, Analysis of full-length tryptophan hydroxylase and the catalyt ic core by molecular sieve chromatography under nondenaturing conditio ns revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consis ting of amino acids 99-444 of the full-length enzyme, contains the seq uence motifs needed for subunit assembly, Both wild-type tryptophan hy droxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron, The tryptophan hydroxylas e catalytic core is also as active as the full-length enzyme, suggesti ng the possibility that the regulatory domain exerts a suppressive eff ect on the catalytic core of tryptophan hydroxylase.