Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

Abstract

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 (BH) 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, K, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH, binding pocket, which is induced by Trp binding.

Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

“…To produce the monoamine neurotransmitters serotonin and dopamine in vitro, hydroxylases specific to the amino acid substrate were exploited. The catalytic domain of human tryptophan hydroxylase isoform 2 (TPH) was chosen for the synthesis of 5‐HTP from Trp, [ 9 ] because this enzyme was previously shown to not be inhibited by high concentrations of the substrate. [ 10 ] Two versions of the catalytic domain of rat tyrosine hydroxylase were tested for the production of L‐DOPA, including a recombinant, wild type version (rTH) and a truncated construct (ΔTH) that was not inhibited by substrate.…”

Characterization of serotonin and N‐acetylserotonin systems in the human epidermis and skin cells

“…Serotonin (5HTP), a product of sequential metabolic transformation of L‐tryptophan (Trp) → hydroxyTrp → 5HTP, is produced across different species representing an ancient molecule with pluripotent and diverse activities . The rate‐limiting step in serotonin synthesis is tryptophan hydroxylation at position 5 on the indole ring to produce 5‐hydroxytryptophan (TrpOH) in a reaction catalyzed by tryptophan hydroxylase and requiring molecular oxygen and the reducing cofactor 6‐tetrahydrobiopterin . There are two isoforms of the enzyme encoded by separate genes.…”

“…There are two isoforms of the enzyme encoded by separate genes. TPH1 expression is the highest in the pineal gland, while being detected in different body organs and cells types including skin, and TPH2 expressed predominantly in neuronal cells and representing the main isoform in the brain . Both isoenzymes are expressed in the human skin with TPH2 being predominantly expressed in normal and malignant melanocytes and retinal pigment epithelium .…”

Towards an Integrative Understanding of tRNA Aminoacylation–Diet–Host–Gut Microbiome Interactions in Neurodegeneration

“…The estimated tryptophan/tryptamine ratio does not reflect the exact in vivo status and depends on the methods of extraction and determination. The TrpRS substrate inhibition by Trp seems plausible because the Trp substrate inhibition was also demonstrated for other human enzymes implicated in tryptophan metabolism, such as Indoleamine 2,3-Dioxygenase [ 38 , 39 ] and Tryptophan Hydroxylase [ 40 ]. Moreover, Trp inhibits an enzyme specified by the tryptophan operon: anthranilate synthetase in bacteria [ 41 ] and plant [ 42 ].…”