BIOCHEMISTRY LAB
FUNCTION OF A COENZYME:
BENZOIN CONDENSATION OF BENZALDEHYDE USING THIAMINE
Coenzymes in biological systems are analogous to “reagents” in laboratory experiments.
Coenzymes usually form a complex with specific enzymes, which subsequently bind the
substrates. The coenzyme reacts with the substrate while they are both bound to the
enzyme. Since most biochemical processes are no more than organic chemical reactions
carried out under special conditions, they can be explained mechanistically, if they have
been studied well enough. Enzyme-substrate-coenzyme complexes make these reactions
more efficient (faster with higher yields), more selective, and more stereoselective (as
well as stereospecific). Furthermore, these reactions occur at milder conditions (pH) than
would normally be possible at laboratory conditions.
Thiamine and its pyrophosphate derivative are coenzymes universally present in all living
systems. Thiamine was originally discovered as a required nutritional factor (vitamin –
like most coenzymes) in humans by its link with the disease beriberi. The deficiency of
Vitamin B1 causes beriberi, which is a disease of the peripheral nervous system.
Symptoms include pain and paralysis of the extremities, emaciation, or swelling of the
body. The disease is most common in locations in the world, where people suffer from
malnutrition.
pyrophosphate
pyrimidine ring
N
O
N
NH 2
O
S
N
P
O
O O
P
O
O
H
thiazole ring
Thiamine pyrophosphate
Thiamine serves as a coenzyme for three important types of enzymatic reactions:
1.
Formations of acyloins (-hydroxy ketones –as in benzoin condensation)
O
O
2 R
thiamine
R
R
H
OH
2.
Nonoxidative decarboxylations of -keto acids
O
R
3.
O
CO2
thiamine
R
H
+
CO2
Oxidative decarboxylations of -keto acids
O
R
O
CO 2
thiamine
O2
R
H
+
CO 2
General Mechanism of thiamine mediated reactions:
The reactive site of thiamine is the thiazole ring which undergoes deprotonation at the
C-2 position under basic conditions to form a stabilized conjugate base. Due to the
charge on the trisubstituted ring nitrogen (pyridine like), the conjugate acid is an
ylide. The sulfur atom plays an important role in stabilizing the ylide.
R'
R
R'
S
N
R
N
S
R
O
H
ylide
R'
R'
H B
HO
H
S
N
R
N
S
HO
H
B
R'
R
N
H O
R'
B H
S
O
R
N
H
R
O
OH
H
O
R'
S
N
S
+
OH
benzoin
Procedure:
Add 1.0 g thiamine hydrochloride to the vial supplied. Dissolve the solid in 1.4 mL of
water by sonicating the vial. Add 10 mL of 95% ethanol and sonicate the solution
until it is homogeneous. To this solution add 3.0 mL of an aqueous sodium hydroxide
solution (2 M). Sonicate the vial until the bright yellow color fades to a pale yellow
color. Carefully measure 3.0 mL of benzaldehyde and add it (with a syringe) to the
flask. Sonicate the vial until it is homogenous. Put the screw top on the flask and
allow it to stand in a dark place until the crystals form. It is likely that this process
will not take place earlier than two-three days.
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Exercises:
1. Propose a mechanism for the non-oxidative decarboxylation of -keto acids.
Given below is the first step between pyruvic acid and thiamine. Show a step by
step mechanism and give the final products.
R'
R
N
S
O
CO 2
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2.
Propose a mechanism for the oxidative decarboxylation of -keto acids. The
substrate is again pyruvic acid; however another cofactor, lipoic acid, is utilized.
Start with the third step shown below to give a step by step outline and the final
products.
R'
R
N
HO
S
H B
S S
CO 2
lipoic acid
3. What makes the above reaction oxidative? In your answer compare the oxidation
states of the products in question 1 and question 2. What is the oxidizing agent in
the reaction as shown above?
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4. How do you think lipoic acid could be attached to an enzyme. Think of an
enzyme where lipoic acid could be covalently attached to the enzyme. Comment
on a possible specific amino acid residue instrumental in the attachment.
5. Do you expect thiamine to be phosphorylated (in its pyrophosphate form) or
unphosphorylated when it forms a complex with an enzyme? Explain.
6. Propose amino acid residues for the active site of the enzyme for question 1.
Consider also protonating species which might be taking part in the process.
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