Studies on the Nature of the Binding of Thiamine Pyrophosphate to Enzymes

作者：Amit Morey, Elliot Juni · 发表于：Journal of Biological Chemistry · 年份：1968 · DOI：10.1016/s0021-9258(18)93372-7 · 被引用次数：46 · 研究领域：Biochemical Acid Research Studies、Alcoholism and Thiamine Deficiency、Biochemical and Molecular Research

Abstract The binding of thiamine pyrophosphate (TPP) to several enzymes has been determined by measuring cofactor-dependent 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 α-acetolactate 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 α-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-filte...