Mechanistic Insights into the Hydrolysis of Organophosphorus Compounds by Paraoxonase-1: Exploring the Limits of Substrate Tolerance in a Promiscuous Enzyme.

We designed, synthesized and screened a library of analogs of the organophosphate pesticide metabolite paraoxon against a recombinant variant of human serum paraoxonase-1. Alterations of both the aryloxy leaving group and the retained alkyl chains of paraoxon analogs resulted in substantial changes to binding and hydrolysis, as measured directly by spectrophotometric methods or in competition experiments with paraoxon. Increases or decreases in the steric bulk of the retained groups generally reduced the rate of hydrolysis, while modifications of the leaving group modulated both binding and turnover. Studies on the hydrolysis of phosphoryl azide analogs as well as amino-modified paraoxon analogs, the former being developed as photo-affinity labels, found enhanced tolerance of structural modifications, when compared with O-alkyl substituted molecules. Results from computational modeling predict a predominant active site binding mode for these molecules which is consistent with several proposed catalytic mechanisms in the literature, and from which a molecular-level explanation of the experimental trends is attempted. Overall, the results of this study suggest that while paraoxonase-1 is a promiscuous enzyme, there are substantial constraints in the active site pocket, which may relate to both the leaving group and the retained portion of paraoxon analogs.

Protein design provides lead(II) ion biosensors for imaging molecular fluxes around red blood cells.

Metalloprotein design and semiconductor nanoparticles have been combined to generate a reagent for selective fluorescence imaging of Pb(2+) ions in the presence red blood cells. A biosensor system based on semiconductor nanoparticles provides the photonic properties for small molecule measurement in and around red blood cells. Metalloprotein design was used to generate a Pb(2+) ion selective receptor from a protein that is structurally homologous to a protein used previously in this biosensing system. Parameters for the Pb(2+) ion binding site were derived from crystallographic structures of low molecular weight Pb(2+) ion complexes that contain a stereoactive lone pair. When the designed protein was produced and attached to ZnS-coated CdSe nanoparticles, two Pb(NO(3))(2)-associated binding events were observed (2-fold emission decrease; K(A1) = 1 x 10(9) M(-1); K(A2) = 3.5 x 10(6) M(-1)). The fluorescence response had a 100 pM Pb(NO(3))(2) detection limit, while no response was observed with Ca(2+) ions (10 mM), Zn(2+) ions (100 muM), or Cd(2+) ions (100 muM). Metal ion selectivity presumably comes from the coordination geometry selected to favor lone pair formation on Pb(2+) ions and electrostatically disfavor tetrahedral coordination. Replacement of ZnS-coated CdSe with ZnS-coated InGaP nanoparticles provided similar biosensors (100 pM limit of detection; K(A1) = 1 x 10(9) M(-1); K(A2) = 1 x 10(7) M(-1)) but with excitation/emission wavelengths longer than the major absorbance of red blood cell hemoglobin (>620 nm). The InGaP nanoparticle-based biosensors provided a 5 nM Pb(NO(3))(2) detection limit in the presence of red blood cells. The modularity of the biosensor system provides exchangeable Pb(2+) ion detection around red blood cells.

Reactivity studies, structural characterization, and thermolysis of cubic titanosiloxanes: precursors to titanosilicate materials which catalyze olefin epoxidation.

The cubic titanosiloxane [RSiO(3)Ti(OPr(i))](4) (R = 2,6-Pr(2)(i)C(6)H(3)NSiMe(3)) (1) is found to be relatively inert in its attempted reactions with alcohols and other acidic hydrogen containing compounds. The reaction of 1 with silanol (Bu(t)O)(3)SiOH however proceeds over a period of approximately 3 months to result in the hydrolysis of (Bu(t)O)(3)SiOH and yield the transesterification product [RSiO(3)Ti(OBu(t))](4) (2) rather than the expected [RSiO(3)Ti(OSi(OBu(t))(3))](4). Products 1 and 2 have been characterized by elemental analysis, thermal analysis, and spectroscopic techniques (IR, EI-MS, and NMR). The solid-state structures of both 1 and 2 have been determined by single-crystal X-ray diffraction studies. Compounds 1 and 2 are isomorphous and crystallize in a cubic space group with a central cubic Ti(4)Si(4)O(12) core. Solid state thermolysis of 1 was carried at 450, 600, 800, 900, 1000, and 1200 degrees C in air, and the resulting titanosilicate materials 1a-f were characterized by spectroscopic (IR and DR UV), powder XRD, and electron microscopic methods. While, the presence of Ti-O-Si linkages appears to be dominant in the samples prepared at lower temperatures (450-800 degrees C), phase separation of anatase and rutile forms of TiO(2) occurs at temperatures above 900 degrees C as revealed by IR spectral and PXRD studies. The presence of octahedral titanium centers was observed by DR UV spectroscopy for the samples heated at higher temperatures. The use of new titanosilicate materials as catalysts for olefin epoxidation has been investigated. The titanosilicate materials produced at temperatures below 800 degrees C with a large number of Ti-O-Si linkages (or tetrahedral titanium centers) were found to be more active catalysts compared to the materials produced above 900 degrees C. The observed conversion in the epoxidation reactions was found to be somewhat low although the selectivity of the epoxide formation over the other possible oxidized products was found to be very good.