| Abstract
| - Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5‘-phosphate (PLP)-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyr to give phenol and ammoniumpyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of thesubstrate, followed by deprotonation of the α-carbon to give a quinonoid intermediate. Elimination ofphenol then has been proposed to give an α-aminoacrylate Schiff base, which releases iminopyruvatethat ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kineticexperiments have provided direct spectroscopic evidence for the formation of the external aldimine andquinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted α-aminoacrylateintermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilicanalogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL withS-alkyl-l-cysteines, l-tyrosine, and 3-fluoro-l-tyrosine. In the presence of 4-hydroxypyridine, a newabsorption band at 338 nm, assigned to the α-aminoacrylate, is observed with these substrates. Formationof the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbesticpoints at ∼365 nm. The value of the rate constant for aminoacrylate formation is similar to kcat, suggestingthat leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-l-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the α-aminoacrylate isobserved, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibitkinetic isotope effects of ∼2−3 on α-aminoacrylate formation when the α-2H-labeled substrates are used,consistent with the previously reported internal return of the α-proton to the phenol product. These resultsare the first direct spectroscopic observation of α-aminoacrylate intermediates in the reactions of TPL.
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