Iodotyrosine
deiodinase (IYD) is unusual in its reliance on flavin
to promote reductive dehalogenation of halotyrosines under aerobic
conditions. Applications of this activity can be envisioned for bioremediation,
but expansion of its specificity requires an understanding of the
mechanistic steps that limit the rate of turnover. Key processes capable
of controlling steady-state turnover have now been evaluated and described
in this study. While proton transfer is necessary for converting the
electron-rich substrate into an electrophilic intermediate suitable
for reduction, kinetic solvent deuterium isotope effects suggest that
this process does not contribute to the overall efficiency of catalysis
under neutral conditions. Similarly, reconstituting IYD with flavin
analogues demonstrates that a change in reduction potential by as
much as 132 mV affects k
cat by less than
3-fold. Furthermore, k
cat/K
m does not correlate with reduction potential and indicates
that electron transfer is also not rate determining. Catalytic efficiency
is most sensitive to the electronic nature of its substrates. Electron-donating
substituents on the ortho position of iodotyrosine
stimulate catalysis and conversely electron-withdrawing substituents
suppress catalysis. Effects on k
cat and k
cat/K
m range from
22- to 100-fold and fit a linear free-energy correlation with a ρ
ranging from −2.1 to −2.8 for human and bacterial IYD.
These values are consistent with a rate-determining process of stabilizing
the electrophilic and nonaromatic intermediate poised for reduction.
Future engineering can now focus on efforts to stabilize this electrophilic
intermediate over a broad series of phenolic substrates that are targeted
for removal from our environment.
Four-component reactions of 3-amino-1,2,4-triazole or 5-amino-1H-pyrazole-4-carbonitrile with aromatic aldehydes and pyruvic acid or its esters under ultrasonication were studied. Unusual for such a reaction type, a cascade of elementary stages led to the formation of 7-azolylaminotetrahydroazolo[1,5-a]pyrimidines.
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