Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase
Kasper D Tidemand1, Günther H Peters1, Pernille Harris1
1Department of Chemistry, Technical University of Denmark , Kemitorvet 207, DK-2800 Kongens Lyngby, Denmark.
Biochemistry
|October 17, 2017
Summary
Tryptophan hydroxylase 1 (TPH1) exhibits unique substrate inhibition due to an active site loop, unlike TPH2. Mutations in this loop alter TPH1 kinetics by affecting the tetrahydrobiopterin binding pocket.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Tryptophan hydroxylase (TPH) is crucial for serotonin biosynthesis, a neurotransmitter implicated in disorders like depression.
- TPH has two isoforms, TPH1 (peripheral) and TPH2 (central), with significant sequence identity.
- Understanding TPH kinetics is vital for developing targeted therapies.
Purpose of the Study:
- To determine the steady-state kinetic mechanism of the human TPH1 catalytic domain.
- To investigate the kinetic differences between TPH1 and TPH2, particularly TPH1's substrate inhibition.
- To elucidate the structural basis for these kinetic differences.
Main Methods:
- Steady-state kinetic assays with varying concentrations of tryptophan (Trp) and tetrahydrobiopterin (BH4).
- Sequence alignment and comparative analysis of TPH1 and TPH2 catalytic domains.
- Site-directed mutagenesis of the active site loop in TPH1.
- Molecular dynamics simulations.
Main Results:
- TPH1 follows a hybrid Ping Pong-ordered mechanism, influenced by tryptophan concentration.
- TPH1 exhibits substrate inhibition by tryptophan, a characteristic absent in TPH2.
- Mutations in TPH1's active site loop significantly alter kinetic parameters, increasing the inhibition constant (Ki).
- Molecular dynamics revealed Trp binding induces pocket closure in the BH4 binding site of TPH1.
Conclusions:
- The active site loop is responsible for the distinct substrate inhibition of TPH1.
- Structural differences in this loop explain the differing kinetic properties between TPH1 and TPH2.
- Targeting this loop could offer a strategy for modulating serotonin synthesis in peripheral tissues.
![Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol](/_next/image?url=https%3A%2F%2Fcloudfront.jove.com%2FCDNSource%2Fteasers%2F63025.jpg&w=3840&q=50)
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