Effects of tyrosine ring fluorination on rates and equilibria of formation of intermediates in the reactions of carbon-carbon lyases.
The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol-lyase and tryptophan indole-lyase (tryptophanase) were studied by rapid-scanning stopped-flow spectrophotometry. The reaction of L-tyrosine with tyrosine phenol-lyase resulted in rapid formation of a small absorbance peak at 500 nm, attributed to a quinonoid intermediate. The reaction of 3-fluoro-L-tyrosine with tyrosine phenol-lyase resulted in a peak at 500 nm with much higher absorbance, as did the reaction of 3,5-difluoro-L-tyrosine, due to increased accumulation of quinonoid intermediates. In constrast, complexes with 2-fluoro-L-tyrosine, 2,3-difluoro-L-tyrosine, 2,5-difluoro-L-tyrosine, and 2,6-difluoro-L-tyrosine exhibited much lower absorbance intensity at 500 nm. The rate constant for quinonoid intermediate formation from 3-fluoro-L-tyrosine was comparable to that for L-tyrosine. However, 3,5-difluoro-L-tyrosine reacted to form a quinonoid intermediate at about half the rate of L-tyrosine, while 2,3-difluoro-L-tyrosine reacted at twice the rate of L-tyrosine. In addition, the 2-substituted difluorotyrosines exhibited an intermediate, which was formed rapidly, absorbing strongly at about 340 nm, which is likely due to a gem-diamine intermediate. Tyrosine is not a substrate for tryptophan indole-lyase; the reaction of tryptophan indole-lyase with L-tyrosine resulted in formation of external aldimine, which absorbed at 420 nm, and a very small absorbance peak at 500 nm. 3-Fluoro-L-tyrosine reacted with tryptophan indole-lyase to produce a prominent quinonoid absorbance peak at 500 nm, whereas L-tyrosine, 2-fluoro-L-tyrosine, and all difluoro-L-tyrosines, had a much reduced intensity for this peak. Thus, the presence of ring fluorine substituents in L-tyrosine that are remote from the site of the chemical transformation has significant effects on the rates and equilibria of intermediate formation in the reactions with both tyrosine phenol-lyase and tryptophan indole-lyase. Although it is commonly thought that fluorine substitution will not result in any significant steric effects, our results suggest that the effects of fluorine substitution in the reactions of fluorinated tyrosines with tyrosine phenol-lyase and tryptophan indole-lyase are due to a combination of steric and electronic effects.
Phillips
R S
RS
Department of Chemistry, University of Georgia, Athens 30602-2556, USA. phillips@bscr.uga.edu
Von Tersch
R L
RL
Secundo
F
F
D016971:Citrobacter freundii / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D013053:Spectrophotometry; D014368:Tryptophanase / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014447:Tyrosine Phenol-Lyase / Q000235:genetics / Q000378:metabolism*
D005461:Fluorine; D014443:Tyrosine; D014368:Tryptophanase; D014447:Tyrosine Phenol-Lyase