| Abstract
| - The binding of small ligands to symmetrical oligomericproteins may lead to a number ofdifferent partially ligated intermediates but should finally yield asymmetrical fully ligated enzyme/ligandcomplex. In the case of the trimeric protein, riboflavin synthase,some ligands form an unexpected protein/ligand complex, even in the presence of a large excess of ligand.Three different bound forms wereobserved by 19F NMR spectroscopy, and Scatchard-typeanalysis suggested binding sites of similar affinities.NOESY analysis of the kinetic network revealed that the threebound states exchange with free ligand,but not with each other, thus suggesting that the trimeric enzyme couldbe asymmetrical. This informationpermits appropriate precautions to be taken during X-ray structureanalysis of riboflavin synthase, whichis in progress. Quantitative analysis of the NOESY spectra yieldeddifferent rate constants for the differentbinding sites. For comparison, the monomeric lumazine protein wasinvestigated as an example of a casewith simple two-site exchange. For such systems, all kineticparameters including kon and thedissociationconstant can be determined from the NOESY spectrum. The data showthat NMR spectroscopy canproduce qualitative and quantitative information in cases ofnonequivalent binding sites in oligomericproteins if isolated NMR signals of the different forms can beobserved. The technique is not limited to19F as reporter nucleus.
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