Hübner G, Weidhase R, Schellenberger A
Eur J Biochem. 1978 Dec 1;92(1):175-81. doi: 10.1111/j.1432-1033.1978.tb12735.x.
The sigmoidal shape of the curve for v[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2-oxo acids but also by 2-oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme-activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is independent of the structure of the activator molecules. The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped-flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data. Using glyoxylic acid as an irreversibly binding active-site marker, it could be shown that addition of the substrate to the enzyme-bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.
丙酮酸脱羧酶的v[S]曲线呈S形，这表明该酶的催化活性受底物调控。该酶在没有底物时无活性，不仅可被2-氧代酸激活，还能被2-氧代酸酰胺激活，而2-氧代酸酰胺不能作为该酶的底物。虽然酶-激活剂复合物的解离常数取决于羰基的亲电性质，但在激活剂物种饱和浓度下达到的催化活性与激活剂分子的结构无关。通过两个可逆步骤进行的激活机制可以用停流技术精确评估。估算了激活和失活反应的动力学参数，通过将推导的描述激活动力学的方程与实测数据进行比较，证明了这些方程的有效性。使用乙醛酸作为不可逆结合的活性位点标记物，可以表明将底物添加到与酶结合的硫胺素二磷酸上是催化机制中的一个步骤，其速率受底物（激活剂）分子控制。
