Binding of phenol and analogues to alanine complexes of tyrosine phenol-lyase from Citrobacter freundii: implications for the mechanisms of alpha,beta-elimination and alanine racemization.
We have examined the interaction of Citrobacter freundii tyrosine phenol-lyase with both L- and D-alanine. This enzyme catalyzes the racemization of alanine as a side reaction, in addition to the physiological beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. The steady-state kinetic parameters for alanine racemization, kcat and Km, for D-alanine are 0.008 S-1 and 32 mM, respectively, while those for L-alanine are 0.03 S-1 and 11 mM. Incubation of tyrosine phenol-lyase with either L- or D-alanine forms a quinonoid complex that exhibits a strong peak at 500 nm. The presence of K+ increases the intensity of the 500-nm absorption with L-alanine, but decreases the intensity of the peak with D-alanine. Rate constants for the formation of these quinonoid intermediates and the effects of phenol and analogues on the reaction with either L- or D-alanine have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Phenol binds to all the intermediates of tyrosine phenol-lyase with L- and D-alanine, but most strongly to the external aldimine complex, resulting in a decrease in the absorbance at 500 nm at equilibrium. Pyridine N-oxide binds selectively to the quinonoid complex of alanine, and thus causes an increase in the absorbance at 500 nm at equilibrium. 4-Hydroxypyridine causes a decrease in absorbance at 500 nm during the fast phase, but an increase in absorbance at 502 nm in a subsequent slow relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
H
H
Department of Chemistry, University of Georgia, Athens 30602.
Phillips
R S
RS
D000409:Alanine / Q000378:metabolism*; D002942:Circular Dichroism; D016971:Citrobacter freundii / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D019800:Phenol; D010636:Phenols / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism; D011728:Pyridones; D013053:Spectrophotometry; D013237:Stereoisomerism; D014447:Tyrosine Phenol-Lyase / Q000378:metabolism*
D010636:Phenols; D011725:Pyridines; D011728:Pyridones; D019800:Phenol; C534143:4-pyridone; C013229:pyridine N-oxide; D014447:Tyrosine Phenol-Lyase; D000409:Alanine