Crystallographic Snapshots of Tyrosine Phenol-lyase Show That Substrate Strain Plays a Role in C-C Bond Cleavage

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the C beta-C gamma bond of L-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon carbon bond. As for most other pyridoxal 5'-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-L-tyrosine followed by flash-cooling. The X-ray structures, determined at similar to 2.0 angstrom resolution, reveal two quinonoid geometries: relaxed in the open and tense in the closed state of the active site. The tense state is characterized by changes in enzyme contacts made with the substrate's phenolic moiety, which result in significantly strained conformation at C beta and C gamma positions. We also captured, at 2.25 angstrom resolution, the X-ray structure for the state just after the substrate's C beta-C gamma bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the alpha-aminoacrylate intermediate with bound phenol. In this state, the enzyme ligand contacts remain almost exactly the same as in the tense quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.