Pressure and Temperature Effects on the Formation of Aminoacrylate Intermediates of Tyrosine Phenol-lyase Demonstrate Reaction Dynamics

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 Schiffs 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 angstrom 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 Delta S+, Delta C-p+, Delta V+, and Delta beta+ 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.