Lexicon #2 - Vitamin B1

Vitamin $B_1$: Thiamine

Vitamin $B_1$ or thiamine is neither synthesized nor stored in significant amounts in the human body. Thus, it is a dietary requirement or an essential vitamin. Thiamine is useful as a cofactor in the body only in its pyrophosphate form, that is, as thiamine pyrophosphate (TPP) or thiamine diphosphate (ThDP). It is interesting to note that the region of this molecule that participates in its biochemical reactions has nothing to do with the added pyrophosphate, instead, it occurs at the thiazolium ring of the thiamine molecule.

So why is the pyrophosphate group added to thiamine? The enzymes that TPP binds to as a cofactor (non-covalently) possess pyrophosphate binding sites. In order, to bind to these enzymes and be biologically useful, it is essential that thiamine is attached to a pyrophosphate group. For a better understanding of TPP enzyme binding refer to this stack exchange page.

Structure of thiamine pyrophosphate

Thiamine consists of a thiazolium ring along with an aminopyrimidine ring (its name comes from combining the two: ‘thi’+'amine). Pyrophosphate is added to the ethyl residue on the thiazolium ring to form thiamine pyrophosphate.

Source: Image from MITOCW lexicon. This image has been annotated to show formation of the ylid form of TPP.

The proton on $C_2$ is acidic due to the presence of the quaternary nitrogen atom. On abstraction of this proton, a dipolar carbanion or ylid (pronounced ‘ill-lid’) is formed. This is the active form of the coenzyme. The carbanion (at position $2$) attacks carbonyl carbons as they possess a partial positive charge. The quaternary nitrogen (at position $3$) acts as an electron sink for the negative charge that develops on this newly bound carbon and thus, carbanions at this position are stabilized. This mechanism facilitates reactions that would otherwise not occur due to the instability of the intermediate carbanion. One such reaction that is made possible by ylid stabilization is the decarboxylation of alpha keto acids.

Decarboxylation of ketoacids

Beta ketoacids

$\beta$-Ketoacids readily undergo decarboxylation as the product formed after the reaction is resonance stabilized.

Alpha ketoacids

$\alpha$-Ketoacids on the other hand are not easy to decarboxylate. The carbanion formed cannot be stabilized as seen in $\beta$-ketoacids. Thus, the reaction does not take place. However, in the presence of the ylid form of TPP, this carbanion is stabilized and decarboxylation occurs. Thus, TPP is used as a cofactor for enzymes that catalyze the decarboxylation of $\alpha$-ketoacids.

The blue arrow in the above figure points at the carbanion that attacks carbonyl carbons.

Making and breaking $C-C$ bonds

Another set of reactions that proceed with a carbanion intermediate are the making and breaking of carbon bonds. As seen before, the ylid form of TPP stabilizes the intermediate carbanion. Note that for TPP to stabilize the carbanion, an electrophilic center must be present for $C_2$ to attack.

Thiamine pyrophosphate can only stabilize the breaking of a $C-C$ bond if one of the carbons of the bond is an electrophilic center.

Enzymes and reactions involving TPP

Pyruvate decarboxylase

This enzyme is not present in humans. It catalyzes alcoholic fermentation in yeast cells. This reaction occurs after glycolysis to regenerate NAD$^+$ so that glycolysis can occur repeatedly in the cell. The pyruvate formed at the end of glycolysis is converted to ethanol. During this reaction, an intermediate product, acetaldehyde is formed. On studying the structure of pyruvate it is easy to see that it is an $\alpha$-keto acid. It undergoes decarboxylation facilitated by the ylid form of TPP to form acetaldehyde.

Source: MITOCW lexicon page 17.

Pyruvate dehydrogenase

The pyruvate dehydrogenase multienzyme complex (PDC) catalyzes the link reaction in aerobic organisms. It converts the end product of glycolysis (pyruvate) into the substrate required for the first reaction of the citric acid cycle (acetyl CoA) thereby linking together these two pathways. The PDC consists of three enzymes which perform five sequential reactions to convert pyruvate to acetyl CoA.

Enzymes of the PDC:
E1= Pyruvate dehydrogenase
E2= Dihydrolipoyl transacetylase
E3= Dihydrolipoyl dehydrogenase

The first enzyme (E1) pyruvate dehydrogenase uses TPP as a cofactor. Its mechanism is similar to that of pyruvate decarboxylase except that it does not convert the hydroxyethyl$-$TPP intermediate into acetaldehyde and TPP. Instead, the hydroxyethyl group is transferred to the next enzyme in the multienzyme sequence, dihydrolipoyl transacetylase (E2). This eventually results in the formation of the oxidized form of lipoic acid as shown in the figure above. For a detailed explanation on the PDC multienzyme complex refer to this blog post.

2-ketoacid dehydrogenases

As we saw above, the pyruvate dehydrogenase complex is a 2-ketoacid dehydrogenase (also know as alpha ketoacid dehydrogenase or 2-oxo-acid dehydrogenase). All the enzymes of this family catalyze the decarboxylation of alpha ketoacids and hence require TPP as a cofactor.
Enzymes of the 2-ketoacid family:

• Pyruvate dehydrogenase
• Alpha ketoglutarate dehydrogenase complex
• Branched-chain alpha keto acid dehydrogenase complex

Enzymes catalyzing alpha ketoacid decarboxylation require TPP as a cofactor.

Transketolase

Transketolase cleaves off the bond between the carbonyl carbon and the rest of the compound. Then, it transfers the this keto group onto another compound. The carbanion of the ylid form of TPP attacks the carbonyl carbon of the substrate. Subsequent carbanion formation and stabilization takes place. Finally, the keto group is cleaved off the enzyme and transferred to another carbonyl carbon.

Transketolase breaks a $C-C$ only if one of the carbons is a carbonyl carbon (electrophilic center). It then transfers this cleaved off keto group onto another carbonyl carbon (electrophilic center).

Transketolase is seen in the carbon scrambling reactions of the pentose phosphate pathway. One of the two reactions it catalyzes is shown below.

Do not confuse transketolase with transaldolase. The mechanism of action and the bonds broken are vastly different. The difference between the two enzymes is explained in this stack exchange answer.

Deficiency of vitamin $B_1$

Unpolished rice is a rich source of thiamine (0.2mg/cup). Polished rice on the other hand contains only 0.039mg of thiamine per cup. Parboiling of rice (partial boiling) causes the rice kernels to absorb nutrients from their outer layers, thereby increasing thiamine content of rice. This thiamine content is retained on polishing. Thiamine pyrophosphate reactions such as the reaction of pyruvate dehydrogenase results in NADH production and thereby ATP production. Thus, thiamine deficiency also results in overall ATP deficiency. Due to their high ATP utilization, the heart and brain are primarily affected in beriberi a condition resulting from thiamine deficiency.

Sources

-Thumbnail: Rice is a rich source of thiamine

• Biochemistry by Donald Voet and Judith G. Voet
• MITOCW course 5.07 Biological Chemistry I: Fall 2013
• MITOCW Lexicon of course 5.07
• Textbook of Biochemistry for Medical Students Book by Damodaran M. Vasudevan and S. Sreekumar