ELUCIDATING THE MECHANISM OF TYROSINE PHENOL-LYASE Alfred Antson¹, Guy Dodson¹, Keith Wilson^1,2 and Tatyana Demidkina³, ¹Department of Chemistry, University of York, York Y01 5DD, UK., ²European Molecular Biology Laboratory (EMBL), c/o DESY, Notkestrasse 85, D-2000 Hamburg 52, Germany, ³Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St., Moscow 117984, Russia

To understand chemical and structural events associated with the catalysis by tyrosine phenol-lyase (TPL) we performed X-ray studies of several complexes of this enzyme with cofactor (pyridoxal 5'-phosphate) and substrate analogues. TPL catalyses reversible ß-elimination of L-tyrosine to produce pyruvate, phenol and ammonia. This reaction goes through a number of intermediate steps which involve two enzyme-associated bases that abstract protons from the substrate Ca and phenol hydroxyl. TPL requires K+ for activity and in addition to physiological reaction catalyses ß-elimination of a number of ß-substituted amino acids and also the racemisation of alanine. In our study we used the X-ray structure of the apo enzyme as a crystallographic matrix for structure solution of different TPL complexes. The monovalent cation binding site has been derived from difference Fourier maps between X-ray data from apo enzyme crystals soaked with Cs+ and K+. The structure! of the holo enzyme, obtained by co-crystallisation with pyridoxal 5'-phosphate, has been refined to a crystallographic R-factor of 17.7 % (Rfree=20.5 %) at 1.9 Å resolution. X-ray data have been collected from freeze-trapped complexes of TPL with L-Alanine and with L-Alanine + 4-hydroxypyridine. The last two complexes were prepared by soaking the holo enzyme crystals and are thought to represent the quinonoid intermediate that forms after the abstraction of the Ca proton. Refinement of these structures is presently underway. Details of the ligand interactions and the possible catalytic mechanism will be presented.