Abstract
Thiamin diphosphate, the biologically active form of vitamin B,, functions as a cofactor in various enzymes in the cell. The protein enhances the reactivity of the cofactor by binding it in a very specific manner. In this work, based upon information from the crystal structure, the mechanism of the thiamin dependent enzyme transketolase from yeast has been investigated by various methods.
In enzymatic thiamin catalysis, the protein has three major tasks in the formation of a functional enzyme. It has to provide a suitable binding pocket for the metal, cofactor and the substrates. It has to stabilise the cofactor in a conformation where the 4'-amino group of the aminopyrimidine ring is positioned orthogonal to and very close in space to the reactive C2 carbon atom of the thiazolium ring. Finally, it has to keep the N1' atom of the aminopyrimidine ring in a protonated state, leading to the conversion of the 4'-amino group to the basic 4'-imino species. This group then abstracts the thiazolium C2 proton to form the reactive carbanion.
The crucial protonation of the aminopyrimidine N1' atom is performed by the conserved residue Glu418. The protein does not stabilise the C2 carbanion. Instead, it speeds up the exchange of the C2 proton to a non-limiting rate. A hydrogen bond network consisting of conserved glutaric acids and water molecules at the subunit interface, connects the two cofactor binding sites. When one of these glutamates (Glu162) is mutated, the formation of the transketolase dimer is perturbed. When bound to the protein, the positively charged thiazolium ring of thiamin diphosphate is stabilised by electrostatic interaction with the conserved Asp382. As the C2 carbanion attacks the substrate, the negative charge of the substrate carbonyl oxygen is stabilised by the charged 4'-imino group of the cofactor and also by interaction with the side chain of His481. The B-carbon hydroxyl group can form hydrogen bonds to His69 and HislO3. In the cleavage of the donor substrate, the conserved His30 orients the substrate C3 hydroxyl group and facilitates proton abstraction by His263. In the subsequent additional step, His263 donates a proton to the aldehyde oxygen of the acceptor substrate.
