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 herbicola 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-dihydroxyphenylalanine (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) values were found to be pH-independent. For 2-chlorotyrosine and DOPA V(max) decreased at lower pH, the effect being described by equation with one pKa. Generally two bases are reflected in the pH dependence of V(max)/Km. The first base, probably is responsible for the abstraction of alpha-proton, while the second one, interacts with the phenolic hydroxyl at the stage of binding. The reaction of TPL with DOPA differs from the reactions with other tyrosines by the requirement of an additional base which is reflected in the pH-profiles of both V(max) and V(max)/Km. For the reaction of TPL from Citrobacter intermedius with DOPA only V(max)/Km values could be determined. The activity of Citrobacter enzyme towards DOPA is considerably less than that of E. herbicola enzyme, and its maximal value is attained at higher pH.
Faleev
N G
NG
Institue of Food Substances, Russian Academy of Sciences, Moscow, Russia.
Spirina
S N
SN
Ivoilov
V S
VS
Demidkina
T V
TV
Phillips
R S
RS
D000645:Ammonium Sulfate; D002850:Chromatography, Gel; D002954:Citrobacter / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004885:Erwinia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011479:Protamines; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D014447:Tyrosine Phenol-Lyase / Q000302:isolation & purification / Q000378:metabolism*
D011479:Protamines; D014443:Tyrosine; D014447:Tyrosine Phenol-Lyase; D000645:Ammonium Sulfate