Expression and Characterization of the Catalytic Core of Tryptophan Hydroxylase

Abstract

Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH 102-416 , was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH 102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.Tryptophan hydroxylase (TRH 1 , EC 1.14.16.4) carries out the 5-hydroxylation of tryptophan via the oxidation of tetrahydropterin and the reductive incorporation of molecular oxygen (Scheme 1). In mammalian metabolism the reaction catalyzed by TRH precedes ␣-decarboxylation and is believed to be the initial and rate-limiting process in the production of the neurotransmitter serotonin (5-hydroxytryptamine). Although TRH has been studied since the early 70s, enzymological characterization has been impeded by the limited quantity of active enzyme available from native or heterologous sources, the exceedingly low specific activity of the isolated enzyme, and the quite rapid decrease in activity observed during purification or storage (1-9).TRH is a member of the small family of pterin-dependent aromatic amino acid hydroxylases that includes tyrosine hydroxylase (TYH) and phenylalanine hydroxylase (PAH). Each of these enzymes catalyzes the addition of an oxygen atom to the ring of an aromatic amino acid substrate. The bulk of what is currently known of the reaction mechanism of these enzymes has come from studies of the latter two (10). Both PAH and TYH require ferrous iron for activity (11,12); however, the exact role for the iron in catalysis is undefined. The primary structures of these enzymes are known from a variety of organisms. Sequence comparisons and deletion mutageneses have identified three functional regions: an amino-terminal regulatory domain, a catalytic domain, and a carboxyl-terminal interface (13-17). The regulatory domains of the three hydroxylases show no similarities, whereas the catalytic domains are homologous, with sequence identities of 32-75%. Enzymes lacking the regulatory domain are catalytically active (13,14,17). The carboxyl-terminal 24 amino acids of TYH form a long helix demonstrated to be responsible for the tetrameric structure of the enzyme (13, 18); this helix is presumed to have a corresponding function in TRH and PAH.We report here the purification and preliminary characterization of a mutant protein containing only the catalytic core of TRH. The rationale for the truncations w...

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

“…In the case of TyrH the latter has been shown to be an Fe(IV) species [16], consistent with the proposed involvement of an Fe(IV)O as the hydroxylating intermediate in all three enzymes [12]. The reactivities of the hydroxylating intermediates in all three enzymes are similar [17] and they have overlapping substrate specificities [18][19][20], supporting the proposal for a common Fe(IV)O intermediate. The iron atom in each is coordinated by two histidines and a glutamate [9][10][11]21,22].…”

“…Moreover, attempts to obtain enzymes containing multiple mutations were not successful (results not shown). The lower yields of the mutant proteins are probably not due to the loss of a stabilizing effect of bound iron, since wild-type versions of the aromatic amino acid hydroxylases can readily be isolated in large amounts even when containing substoichiometric iron or in the apo form [20,[27][28][29]. Instead it suggests that interactions of the side chains of the metal ligands with other residues in the protein are important for structural integrity.…”

Mechanism of Aromatic Amino Acid Hydroxylation

“…For TrpH, the V/K value for tryptophan is only 12-fold that for phenylalanine, but the enzyme shows a preference for tryptophan over tyrosine of at least 5000-fold (6). TyrH is the least specific of the three enzymes, in that the V/K value for tyrosine is only 10-fold that for phenylalanine (50) and 30-fold that for tryptophan (52).…”

Reduction and Oxidation of the Active Site Iron in Tyrosine Hydroxylase: Kinetics and Specificity

“…The need for the iron in TyrH and the other pterin-dependent amino acid hydroxylases to be in the ferrous form for activity is well established (4,10,11,23). Both TyrH and PheH as isolated contain ferric iron (24)(25)(26), and in both cases the iron can be reduced by tetrahydropterins and other reductants (2,26), with a stoichiometry of 0.5 tetrahydropterin per iron (2,10).…”