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Publications (3)22.13 Total impact

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    ABSTRACT: The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms. Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions. Diffraction data sets to 2.8 A resolution (native) and 2. 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.
    Acta Crystallographica Section D Biological Crystallography 06/1998; 54(Pt 3):467-9. · 14.10 Impact Factor
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    ABSTRACT: The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism. Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase. The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation. In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken. A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced. The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine. The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics. These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine. Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites. Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant. The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites. A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS)
    Biochemistry 08/1995; 34(29):9403-12. · 3.38 Impact Factor
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    ABSTRACT: Control of the regulatory enzyme threonine deaminase from Escherichia coli is achieved by isoleucine inhibition and valine activation. The mechanism by which these heterotropic effectors regulate the enzyme was investigated by measuring the binding of isoleucine and valine by spectroscopic, kinetic, calorimetric and equilibrium dialysis techniques. The addition of isoleucine or valine to threonine deaminase resulted in large changes in the intrinsic fluorescence of the two tryptophans per polypeptide chain. Slightly cooperative binding isotherms for isoleucine were obtained in potassium phosphate, pH 7.5, yielding an average dissociation constant of 4.91 microM, which was confirmed by equilibrium dialysis measurements. Valine binding was much more cooperative, and yielded an average dissociation constant of 122 microM. Titration calorimetry experiments indicated that cooperative heterotropic ligand binding was exothermic, and yielded a stoichiometry of four isoleucine bound per tetrameric enzyme, with an average enthalpy of -10.70 kcal/mol. Valine also bound to four sites per tetramer, with an average enthalpy of -7.45 kcal/mol. The effect of ligands on the fluorescence and circular dichroism spectra of the essential pyridoxal phosphate cofactor indicates that isoleucine and valine bind to effector sites that are distinct from the active sites in threonine deaminase. Shifts in the kinetic properties of threonine deaminase promoted by isoleucine and valine binding are to a first approximation consistent with analyses of effector binding isotherms in terms of a simple two-state model, and suggest that isoleucine regulates threonine deaminase by preferentially binding to the low activity T state, whereas valine binds preferentially to the high activity R state. Finally, analyses of heterotropic effector binding isotherms suggest that active site ligands may have significant affinity for the regulatory sites, which gives rise to underestimates for the allosteric equilibrium constants determined from substrate analog binding isotherms.
    Journal of Biological Chemistry 12/1994; 269(47):29423-9. · 4.65 Impact Factor