Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase.
The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)
Kiick
D M
DM
Department of Biochemistry, University of Wisconsin, Madison 53706.
Phillips
R S
RS
D000409:Alanine / Q000378:metabolism; D001667:Binding, Competitive; D002954:Citrobacter / Q000201:enzymology*; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D003903:Deuterium; D004885:Erwinia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000378:metabolism*
D014443:Tyrosine; D010649:Phenylalanine; D011732:Pyridoxal Phosphate; C008425:S-methylcysteine; D003903:Deuterium; D008190:Lyases; D014447:Tyrosine Phenol-Lyase; D003545:Cysteine; D000409:Alanine