Abstract
The structures of aminoacrylate intermediates of wild-type, F448A mutant, and perdeuterated tyrosine phenol-lyase (TPL) formed from l-tyrosine, 3-F-l-tyrosine, S-ethyl-l-cysteine, and l-serine, with bound 4-hydroxypyridine, were determined by X-ray crystallography. All the aminoacrylate Schiff’s base structures in chain A are identical regardless of the substrate used to form them. 4-Hydroxypyridine is also in an identical location, except for F448A TPL, where it is displaced about 1 Å due to the increased size of the active site. In chain B, we have found different complexes depending on the substrate. With wild-type TPL, l-tyrosine gave no density, 3-F-l-tyrosine gave a gem-diamine, and l-serine gave a gem-diamine in chain B. S-Ethyl-l-cysteine formed an aminoacrylate in chain B with both wild-type and F448A TPL, but perdeuterated TPL with S-ethyl-l-cysteine formed a gem-diamine of aminoacrylate. The kinetics of aminoacrylate intermediate formation from l-tyrosine and S-ethyl-l-cysteine were followed by stopped-flow spectrophotometry at temperatures from 281 to 320 K and hydrostatic pressures ranging from 1 bar to 1.5 kbar at 293 K. There are large negative values of ΔS‡, ΔCp‡, ΔV‡, and Δβ‡ for aminoacrylate intermediate formation for l-tyrosine but not for S-ethyl-l-cysteine. Formation of the aminoacrylate intermediates from l-tyrosine and S-ethyl-l-cysteine shows heavy enzyme deuterium kinetic isotope effects with perdeuterated TPL that are strongly temperature- and pressure-dependent and may be normal or inverse depending on conditions. These results suggest that conformational dynamics as well as vibrational coupling play a key role in the mechanism of the elimination reaction of l-tyrosine catalyzed by TPL.
