MECHANISM OF CATALYSIS BY TYROSINE PHENOL LYASE FROM ERWINIA-HERBICOLA - MULTIPLE KINETIC ISOTOPE EFFECTS FOR THE REACTIONS WITH ADEQUATE SUBSTRATES

The generally accepted mechanism of catalysis by tyrosine phenol lyase (TPL) includes three principal chemical transformations of the substr ate: (1) abstraction of the a-proton in the external aldimine; (2) tau tomerization of the aromatic moiety to convert it into a good leaving group and (3) beta-elimination of the leaving group, The relative sign ificance of these stages has been elucidated for the reactions of TPL from Erwinia herbicola with suitable substrates, L-tyrosine and S-fluo ro-L-tyrosine. The three stages are susceptible to different kinetic i sotope effects (KIEs), To determine the respective KIEs the kinetics o f TPL reactions with normal and alpha-deuteriated 3-fluorotyrosine in water and in (H2O)-H-2, and with beta,beta-dideuteriated substrate in water,were examined, The KIE values that were found (alpha-KIE = 3.4 i n water and 2.0 in (H2O)-H-2; solvent KIE = 1.7; beta-KIE = 1.1) are i n good agreement with the classic stepwise (not concerted) mechanism o f alpha-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 t hat the tautomerization stage is at equilibrium, A reaction mechanism is suggested where the tautomerization stage does contribute significa ntly to the total free-energy barrier although the highest maximum on the free-energy profile corresponds to the subsequent beta-elimination stage.