The binding of thiamine pyrophosphate (TPP) to several enzymes has been determined by measuring cofactordependent activity after passage of each enzyme through a column of Sephadex G-25 to remove non-protein-bound cofactors. TPP was found to be bound irreversibly to yeast and Zymomonas pyruvate decarboxylases, Aerobacter (Yacetolactate synthetase, and Escherichia glyoxylate carboligase. Cofactors were lost when Proteus pyruvate oxidase, Escherichia pyruvate dehydrogenase, and Micrococcus diacetyl carboligase were gel-filtered; the binding of TPP was strongest for diacetyl carboligase. A procedure has been devised for efficient resolution of enzymes for TPP and divalent cations. Resolved enzymes reconstituted for cofactors had properties similar to those of native enzymes. Resolved pyruvate decarboxylases from both yeast and Zymomonas mobilis failed to bind Mg++ in the absence of TPP. Cofactor reconstitution for yeast pyruvate decarboxylase was shown to be a slow process for low concentrations of TPP. TPP alone, in high concentrations, was able to activate and partially reconstitute TPP enzymes in the absence of added divalent cations. Zymomonas pyruvate decarboxylase, Aerobacter a-acetolactate synthetase, and Escherichia glyoxylate carboligase appear to be heterogeneous, in that part of each enzyme can bind TPP irreversibly; this cofactor dissociates reversibly for the remainder of the enzyme. When yeast pyruvate decarboxylase, saturated for cofactors, was gel-filtered at pH 8.0, 50% of the enzyme bound TPP dissociated from the enzyme, the remainder being irreversibly bound. ?Thiazole pyrophosphate,? a potent inhibitor of resolved yeast pyruvate decarboxylase, acts by binding to coenzyme sites on the enzyme. Unlike TPP, thiazole pyrophosphate was shown to be bound reversibly to the enzyme and could be displaced by high concentrations of TPP. The results obtained question the validity of calculating dissociation constants for cofactors which do not dissociate from their enzymes.

• PMID: 4968184
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Journal Article

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Morey AV, Juni E

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