Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization

Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH₄) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, <i>K</i>_<i>i</i>, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH₄, binding pocket, which is induced by Trp binding

Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the Fe(II) active site to form a highly reactive Fe(IV) = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-Fe(IV) = O intermediate and characterized its structure as a Fe(II)-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate–bound ternary Fe(II) active site before the O(2) reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of Fe(IV) = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the Fe(II) site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the Fe(II) site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes