The enzymatic synthesis of thiamin triphosphate.

Abstract

Publisher Summary This chapter describes the enzymatic synthesis of thiamin triphosphate (ThTP). ThTP has been implicated in Leigh's disease (subacute necrotizing encephalomyelopathy) and considerable evidence, albeit circumstantial, suggests a role of this ester in conduction and transmission. The substrate for phosphoryltransferase; thiamin-diphosphate kinase is not free thiamin pyrophosphate (ThPP) but ThPP that is bound to a protein. This substrate is prepared by injecting rats with [ 35 S]thiamin and subsequently isolating the [ 35 S]ThPP-protein from liver by ion-exchange and gel chromatography. After incubation of the labeled substrate with the enzyme and cofactor, [ 35 S]ThTP is isolated by column chromatography and the radioactivity determined. The three active fractions (El-l, El-2, and E2) on disc gel electrophoresis showed a single protein band with the same R f : the molecular weight of the enzyme is calculated to be 103,000. The pH optimum is 7.5, with Tris-HCl exhibiting slightly higher activity than phosphate buffer. The reaction has an absolute dependence on adenosine triphosphate (ATP), Mg 2+ , the cofactor (presumably glucose), and ThPP that is protein bound. When the bound substrate is replaced by free ThPP, no synthesis of ThTP is observed.