COENZYME EXCHANGE BETWEEN HOLOTRANSKETOLASE AND THE MEDIUM

Dihydroxyethylthiamine pyrophosphate (DTPP), a transketolase reaction intermediate, when added exogenously is able to interact with both apo - and holotransketolase. The spectral properties of the complex formed on addition of TPP to holotransketolase differ drastically from those of the complex formed on addition of DTPP to apotransketolase but in essence are similar to those of the naturally occurring DTPP-transketo lase formed in the course of the transketolase reaction, i.e., when th e substrate is mixed with the holoenzyme. DTPP is able to function as substrate in the transketolase reaction even in the presence of the ca talytic amounts of the holoenzyme. The experimental findings suggest t hat DTPP can displace the coenzyme (thiamine pyrophosphate) from holot ransketolase by binding to the enzyme active center already preformed on binding of thiamine pyrophosphate. Deprotonation of DTPP is not the rate-limiting step in the transketolase reaction. There are two indep endent pathways via which holotransketolase-bound thiamine pyrophospha te is exchanged with the medium.