Substrate Analogs Implicate a Free Radical Pathway in Tyrosine Hydroxylase Catalysis

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Heme-dependent tyrosine hydroxylases (TyrH) are vital enzymes in catecholamine biosynthesis.
• The precise role of the substrate's α-amino group in TyrH's monooxygenation mechanism remained unclear.

Purpose of the Study:

• To elucidate the role of the α-amino group in the catalytic mechanism of heme-dependent tyrosine hydroxylases.
• To investigate the reaction pathway of an l-tyrosine analog lacking the α-amino group.

Main Methods:

• Utilized 3-(4-hydroxyphenyl)propionic acid (HPPA), an l-tyrosine analog lacking the α-amino group.
• Employed free radical scavengers to inhibit dimerization.
• Conducted 18O-labeling experiments to track oxygen transfer.
• Performed Electron Paramagnetic Resonance (EPR) spectroscopy with nitrosobenzene.

Main Results:

• Observed a competing dimerization pathway for HPPA, indicative of a radical intermediate.
• Radical scavenger selectively inhibited dimer formation.
• 18O-labeling revealed phenolic oxygen scrambling, suggesting disrupted aromaticity.
• EPR spectroscopy confirmed a substrate-based free radical intermediate.

Conclusions:

• The absence of the α-amino group creates a kinetic bottleneck, enabling radical formation and off-pathway dimerization.
• The native substrate's α-amino group acts as a kinetic modulator, directing the radical towards productive hydroxylation.
• TyrH can operate via a peroxidase-like free radical pathway, with the amino group significantly influencing reaction outcomes.