Mechanism of catalysis by tyrosine phenol lyase from Erwinia herbicola. Multiple kinetic isotope effects for the reactions with adequate substrates

Abstract

The generally accepted mechanism of catalysis by tyrosine phenol lyase (TPL) includes three principal chemical transformations of the substrate: (1) abstraction of the α-proton in the external aldimine; (2) tautomerization of the aromatic moiety to convert it into a good leaving group and (3)β-elimination of the leaving group. The relative significance of these stages has been elucidated for the reactions of TPL from Erwinia herbicola with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine. The three stages are susceptible to different kinetic isotope effects (KIEs). To determine the respective KIEs the kinetics of TPL reactions with normal and α-deuteriated 3-fluorotyrosine in water and in 2H2O, and with β,β-dideuteriated substrate in water, were examined. The KIE values that were found (α-KIE = 3.4 in water and 2.0 in 2H2O; solvent KIE = 1.7; β-KIE = 1.1) are in good agreement with the classic stepwise (not concerted) mechanism of α-proton transfer to the leaving group. The solvent KIEs for the reactions of 3-fluorotyrosine and tyrosine are the same. This result and the low absolute values of the solvent KIEs allow the assumption that the tautomerization stage is at equilibrium. A reaction mechanism is suggested where the tautomerization stage does contribute significantly to the total free-energy barrier although the highest maximum on the free-energy profile corresponds to the subsequent β-elimination stage.