Pressure and temperature effects on formation of aminoacrylate intermediates of tyrosine phenol-lyase demonstrate reaction dynamics

Robert S Phillips, Steven Craig, Andrey Y. Kovalevsky, Oksana O. Gerlits, Kevin L. Weiss, Andreea Iulia Iorgu, Derren J. Heyes, Sam Hay

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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 4-hydroxpyridine bound, 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. The 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 show 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.
Original languageEnglish
JournalACS Catalysis
Early online date19 Dec 2019
Publication statusPublished - 2020

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology


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