Graduate Thesis Or Dissertation
 

The influence of monovalent cations on tryptophanase and pyruvate kinase as studied by fluorescence, circular dichroism, and kinetic methods

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  • Two spectrally different species of holotryptophanase were obtained from fluorescence and circular dichroism measurements. One is predominant in solutions containing certain enzyme activating monovalent cations while the other predominates in solutions of various inhibiting monovalent cations. Relative magnitudes of the cation sensitive fluorescent intensity maxima or circular dichroism maxima are approximately proportional to the effectiveness of the monovalent cations as activators or inhibitors of tryptophanase activity. The tryptophanase coenzyme, pyridoxal 5'-phosphate, is required in order to observe any monovalent cation effects on the enzyme's fluorescence or circular dichroism spectra. Fluorometric titrations indicated that the coenzyme binds to apotryptophanase more tightly in the presence of K ⁺ or NH⁺ ₄ than in Na ⁺ solutions. Similarly, low temperature circular dichroism measurements of the holoenzyme indicated that the coenzyme dissociated more readily in Na⁺ than in K⁺ systems. Activating and inhibiting monovalent cations had essentially similar effects on the binding of the substrate analog, 1,N ⁶-ethenoadenosine diphosphate (εADP), to pyruvate kinase. Likewise, no differential cation affects were observed for the binding of indole to apotryptophanase. Cation effects on the fluorescence anisotropy of tryptophan residues was found to be negligable for all enzymes tested except pyruvate kinase. Addition of activating as well as inhibiting cations to solutions of pyruvate kinase resulted in nearly identical increases in tryptophan anisotropy. It was demonstrated that the anisotropy increase was not caused by a decrease in energy transfer between tryptophan residues. Monovalent cation binding site models are discussed. Evidence in support of allosteric models is lacking. The current results as well as recent work reported in the literature support a functional role for the activating monovalent cation at the pyruvate kinase or tryptophanase active site.
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