Coenzyme binding in yeast pyruvate decarboxylase. A fluorescent enzyme-inhibitor complex.

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The formation of a fluorescent complex between apopyruvate decarboxylase of Saccharomyces carlsbergensis and thiochrome pyrophosphate, a competitive inhibitor with thiamine pyrophosphate, and Mg(II) ions is reported. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate group of the coenzyme in binding to the protein. Complex formation between the apoenzyme and thiochrome pyrophosphate in the absence of Mg(II) could also not be demonstrated. The role of magnesium in coenzyme binding is discussed, especially in reference to interaction with the pyrophosphate moiety. The apparent Km for Mg(II) ions, after correction for residual apoenzyme activity, is reported to be 87 μM.The shift in the fluorescence emission spectrum of thiochrome pyrophosphate of 23–24 nm toward lower wavelengths, upon complex formation, coincided with the behavior of thiochrome in solvents of lower polarity than water. A hydrophobic region at the coenzyme binding site of pyruvate decarboxylase is proposed. Enhancement of thiochrome fluorescence occurred concomitantly with the blue shift in the emission spectrum upon complex formation.The excitation wavelength of 290 nm employed to demonstrate complex formation suggested energy transfer from the tryptophan to the thiochrome chromophore. Quenching of fluorescence in the 340–350 nm region, however, occurred not only in the protein emission spectrum upon complex formation, but also when free tryptophan and thiochrome interacted with each other. The quenching of the protein fluorescence appears to be independent of hydrophobic interaction at the coenzyme binding site. It is suggested that protein tryptophan residues relatively exposed to solvent interact individually with the thiochrome molecule by an energy transfer mechanism to produce the quenching in pyruvate decarboxylase.