5 research outputs found
Rapid Kinetics of Dehalogenation Promoted by Iodotyrosine Deiodinase from Human Thyroid
Reductive dehalogenation such as
that catalyzed by iodotyrosine
deiodinase (IYD) is highly unusual in aerobic organisms but necessary
for iodide salvage from iodotyrosine generated during thyroxine biosynthesis.
Equally unusual is the dependence of this process on flavin. Rapid
kinetics have now been used to define the basic processes involved
in IYD catalysis. Time-dependent quenching of flavin fluorescence
was used to monitor halotyrosine association to IYD. The substrates
chloro-, bromo-, and iodotyrosine bound with similar rate constants
(<i>k</i><sub>on</sub>) ranging from 1.3 × 10<sup>6</sup> to 1.9 × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup>. Only the inert substrate analogue fluorotyrosine exhibited a significantly
(5-fold) slower <i>k</i><sub>on</sub> (0.3 × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup>). All data fit
a standard two-state model and indicated that no intermediate complex
accumulated during closure of the active site lid induced by substrate.
Subsequent halide elimination does not appear to limit reactions of
bromo- and iodotyrosine since both fully oxidized the reduced enzyme
with nearly equivalent second-order rate constants (7.3 × 10<sup>3</sup> and 8.6 × 10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup>, respectively) despite the differing strength of
their carbon–halogen bonds. In contrast to these substrates,
chlorotyrosine reacted with the reduced enzyme approximately 20-fold
more slowly and revealed a spectral intermediate that formed at approximately
the same rate as the bromo- and iodotyrosine reactions
Design of HIV Coreceptor Derived Peptides That Inhibit Viral Entry at Submicromolar Concentrations
HIV/AIDS
continues to pose an enormous burden on global health.
Current HIV therapeutics include inhibitors that target the enzymes
HIV protease, reverse transcriptase, and integrase, along with viral
entry inhibitors that block the initial steps of HIV infection by
preventing membrane fusion or virus–coreceptor interactions.
With regard to the latter, peptides derived from the HIV coreceptor
CCR5 were previously shown to modestly inhibit entry of CCR5-tropic
HIV strains, with a peptide containing residues 178–191 of
the second extracellular loop (peptide <b>2C</b>) showing the
strongest inhibition. Here we use an iterative approach of amino acid
scanning at positions shown to be important for binding the HIV envelope,
and recombining favorable substitutions to greatly improve the potency
of <b>2C</b>. The most potent candidate peptides gain neutralization
breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than
CCR5-tropic strains only. We found that gains in potency in the absence
of toxicity were highly dependent on amino acid position and residue
type. Using virion capture assays we show that <b>2C</b> and
the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies
whose epitopes are located in or around variable loop 3 (V3) on gp120.
Analysis of antibody binding data indicates that interactions between
CCR5 ECL2-derived peptides and gp120 are localized around the base
and stem of V3 more than the tip. In the absence of a high-resolution
structure of gp120 bound to coreceptor CCR5, these findings may facilitate
structural studies of CCR5 surrogates, design of peptidomimetics with
increased potency, or use as functional probes for further study of
HIV-1 gp120–coreceptor interactions