Substrate Analogs Implicate a Free Radical Pathway in Tyrosine Hydroxylase Catalysis

Heme-dependent tyrosine
hydroxylases (TyrH) are critical
enzymes
in catecholamine biosynthesis, yet the role of the substrate’s
α-amino group in their monooxygenation mechanism has been unclear.
Using 3-(4-hydroxyphenyl)propionic acid (HPPA), an l-tyrosine
analog lacking the α-amino group, we observed a distinct dimerization
pathway that competes with the expected hydroxylation reaction. Several
lines of evidence confirm that this process originates from a radical
intermediate. First, the formation of this (HPPA)2 dimer
is selectively inhibited by a free radical scavenger. Second, 18O-labeling experiments show phenolic oxygen scrambling, indicating
a disruption of substrate aromaticity during catalysis. Finally, EPR
spectroscopy using nitrosobenzene as a substrate analog revealed a
substrate-based free radical. This mechanistic divergence clarifies
the role of the α-amino group. Its absence in HPPA creates a
kinetic bottleneck for the final O atom transfer step, allowing a
fraction of the substrate radical to form the off-pathway dimer. Thus,
the native substrate’s α-amino group acts as a crucial
kinetic modulator, ensuring the rapid and efficient commitment of
the substrate radical to productive hydroxylation. These results collectively
establish a peroxidase-like free radical pathway for TyrH and reveal
the nonessential yet significant role the amino group plays in controlling
reaction outcomes.