Ronald Kluger

Lessons from thiamin-watching

Thiamin is a vitamin that is a catalyst for reactions involving formation of acyl carbanion equivalents. The closely related species, thiamin diphosphate, is associated with enzymes that catalyze the decarboxylation of a-ketoacids. While the enzymes attract the ketoacid, thiamin diphosphate adds to it, leading to release of carbon dioxide. Intermediates in the reactions can be synthesized. Kinetic analysis of their reactions reveals what the enzyme overcomes in each step. In the laboratory, thiamin reacts with benzaldehyde to cause a benzoin condensation. In neutral solution, benzaldehyde destroys thiamin.. Analysis of the products and kinetics of destruction reveals the steps by which it occurs: addition and proton transfers followed by a fragmentation step that is itself a mechanistic puzzle.

REACTIVE INTERMEDIATES IN THIAMIN CATALYSIS

The mechanism that Breslow proposed to explain the catalytic function of thiamin in the decarboxylation of pyruvate was based on convincing experimental evidence from work on model reactions (FIGURE l).’,’ The ionization of thiamin at the C-2 position of the thiazolium ring produces an ylid, which serves as a nucleophile toward the carbonyl group of pyruvate, forming a covalent adduct. This adduct has chemical properties that permit C 0 2 to leave and be replaced by a proton much more quickly than if pyruvate were to await its fate independently. The resultant thiamin adduct, 2-a-hydroxyethylthiamin (HET), undergoes further reactions that lead to the product, a ~ e t o i n . ~ By analogy, pyruvate decarboxylase promotes formation of a similar adduct between thiamin diphosphate and pyruvate. Decarboxylation produces the 2-a-hydroxyethyl compound (HET) that the enzyme converts to acetaldehyde, regenerating thiamin diphosphate. The synthesis of HET by Miller el al.‘ and the reaction of HET with acetaldehyde to produce acetoin dramatically confirmed the Breslow mechanism. The isolation of 2-a-hydroxyethylthiamin diphosphate (HETDP) from pyruvate decarboxylase and the demonstration of its ability to generate acetaldehyde with an apoenzyme supported the idea of a common mechanism for enzymic and nonenzymic catalys~s.’.~ A mechanism is, of course, very useful for rationalizing known phenomena. It is also important for making predictions and directing further experimental work by suggesting what questions can be attacked. A whole new area of mechanistic investigation within the Breslow mechanism thus was opened for thiamin researchers. Many important pieces of research have since appeared, as have new hypotheses that elaborate upon this mechanism. The relationship of the enzymic and nonenzymic mechanisms is an intriguing problem. What advantages does the combination of protein and coenzyme have over thiamin alone? In order to answer this we need a quantitative basis for comparisons. Of course we can generalize by comparing the overall rates of enzymic and nonenzymic processes and consider known functions of enzymes. However, since in the case of pyruvate decarboxylation the processes are assumed to involve a similar sequence of steps, we, in principle, have the opportunity of comparing steps. We thus undertook to measure first the kinetics of the nonenzymic reaction. For any study of this sort, it is most efficient to begin with the central intermediate. The pyruvate-thiamin adduct, a-lactylthiamin (LT), occupies the