THE CATALYTIC MECHANISM OF TYROSINE PHENOL-LYASE FROM ERWINIA-HERBICOLA - THE EFFECT OF SUBSTRATE STRUCTURE ON PH-DEPENDENCE OF KINETIC-PARAMETERS IN THE REACTIONS WITH RING-SUBSTITUTED TYROSINES

Apparently homogeneous tyrosine phenol-lyase (TPL) from Erwinia herbic ola has been prepared by a new method. The pH-dependencies of the main kinetic parameters for the reactions of Erwinia TPL with tyrosine, 2- fluorotyrosine, 3-fluorotyrosine, 2-chlorotyrosine, and 3,4-dihydroxyp henylalanine (DOPA) have been studied. The pattern of pH-dependence of V-max depends on the nature of the substituent in the aromatic ring. For the substrates bearing small substituents (H, 2-F, 3-F) V-max valu es were found to be pH-independent. For 2-chlorotyrosine and DOPA V-ma x decreased at lower pH, the effect being described by equation with o ne pK(a). Generally two bases are reflected in the pH dependence of V- max/K-m. The first base, probably is responsible for the abstraction o f alpha-proton, while the second one, interacts with the phenolic hydr oxyl at the stage of binding. The reaction of TPL with DOPA differs fr om the reactions with other tyrosines by the requirement of an additio nal base which is reflected in the pH-profiles of both V-max and V-max /K-m. For the reaction of TPL from Citrobacter intermedius with DOPA o nly V-max/K-m values could be determined. The activity of Citrobacter enzyme towards DOPA is considerably less than that of E. herbicola enz yme, and its maximal value is attained at higher pH.