# Investigating Coenzyme Function of Thiamine Triphosphate Using Its Novel Hydrolysis-Resistant Analog and Transketolase

## TL;DR

Researchers studied the coenzyme function of thiamine triphosphate (ThTP) using a stable analog to determine its role in transketolase activity.

## Contribution

A hydrolysis-resistant analog of ThTP was synthesized to study its coenzyme function without interference from hydrolysis.

## Key findings

- bmThTP acts as a coenzyme for transketolase with an apparent Km of 16.3 µM.
- bmThTP binding differs slightly from ThTP, resulting in a 2.36 kcal/mol ΔG difference.
- ThTP could function as the main coenzyme for transketolase in certain animal species when accumulated.

## Abstract

Thiamine (vitamin B1) and its phosphates are essential for almost all organisms. Thiamine diphosphate (ThDP) is the major intracellular derivative which is considered the only form functioning as a coenzyme. Thiamine triphosphate (ThTP), another ubiquitous derivative, lacks a clear physiological function and is usually kept at low levels. However, it can accumulate up to 87% of total thiamine in animal tissues lacking cytosolic thiamine triphosphatase (THTPA) activity. Studies of ThTP coenzyme function have always faced the problem of ThTP hydrolysis to ThDP. To avoid such interference a synthetic stable ThTP analog, bismethylene ThTP (bmThTP), has been synthesized. Given that ThTP accumulation is caused by cytosolic THTPA suppression, cytosolic ThDP-dependent transketolase (TKT) is the primary target for probing (bm)ThTP’s coenzyme function. Indeed, bmThTP acts as a TKT coenzyme, with the apparent Km(bmThTP) of 16.3 µM. However, bmThTP binding slightly differs from that of ThTP. Molecular docking was used to estimate affinities of ThDP, ThTP and bmThTP, also allowing us to avoid ThTP hydrolysis. Despite almost identical localization within the active site, bmThTP could not bind as well as ThTP, resulting in a 2.36 kcal/mol difference in estimated ΔG. Based on our data, calculated Km(ThTP) for TKT is about 0.07–0.08 µM, only 1.6–2 times that of Km(ThDP). Such a small difference implies that ThTP could physiologically act as the main TKT coenzyme form upon its accumulation in muscles, at least in a few known animal species.