CONTENT

1. INTRODUCTION

2. MECHANISM OF ENZYME ACTION

• FISCHER’S TEMPLATE (OR) LOCK AND KEY MODEL

• KOSHLAND’S INDUCED FIT MODEL

3. ENZYME KINETICS

4. FACTORS AFFECTING ENZYME ACTIVITY

5. ENZYME INHIBITION

6. ROLE OF ENZYMES AND COENZYMES IN METABOLISM

❖ HYDROGEN TRANSFERRING CO-ENZYMES

• NICOTINAMIDE NUCLEOTIDES
• FLAVIN NUCLEOTIDES
• LIPOIC ACID
❖ GROUP TRANSFERRING COENZYMES
• BIOTIN
• THIAMINE DIPHOSPHATE
• PYRIDOXAL PHOSPHATE
• CO-ENZYME-A
• TETRAHYDROFOLATE
• COBAMIDE COENZYME
• ADENOSINE TRIPHOSPHATE
• LIPOAMIDE
• UBIQUINONE
• PROTEIN COENZYME
• CYTOCHROMES

7. REFERENCES

1
 1. INTRODUCTION
Enzymes are the proteins that catalyze biochemical reactions. Study of these important
biochemical reactions was started many years ago, from the time of Louis Pasteur, who for the
first time demonstrated the fermentation of glucose by yeast. The catalytic agent of yeast cell
was subsequently identified and named as ferment. At the end of nineteenth century, a cell-free
extract of yeast was found to be capable of fermenting glucose by Buchner brothers.
Considerable advances were made since then in order to properly know the nature of these
agents named as enzymes and finally their true nature was revealed by James Sumner in 1926
after the extraction and crystallization of the enzyme urease from jack beans. At present, no
less than 150 enzymes have been prepared in crystalline form.

An enzyme can be a large protein made up of several hundred amino acids, or

several polypeptides that act together as a unit. Enzymes have molecular weights ranging from
10,000 to 2,000,000.

Proteins Structure
Until recently, it was understood that all enzymes are proteins. However, observations
made in organelles from plants, yeast, viruses and higher eukaryotic cells, show that RNA can
act as an enzyme. Such RNA molecule is called a ribozyme. Hence, ribozymes are RNA
molecules with catalytic activity. These generally involve transesterification reactions, and
most are concerned with RNA metabolism (splicing and endoribonuclease). Recently, a
ribosomal RNA component was noted to hydrolyze an aminoacyl ester and thus to play a
central role in peptide bond function i.e. having peptidyl transferase activity.

2
 Definitions of terms related to enzymes

Cofactor Organic molecules or ions that assist many enzymes in their reactions.

Apoenzyme The protein portion of an enzyme requiring a cofactor for its reaction.

Holoenzyme A whole enzyme, as a complete and functional molecule. Generally, a

holoenzyme consists of a polypeptide portion (an apoenzyme) and at least
one cofactor or another coenzyme.

Enzyme Proteins that act as catalysts, speeding the rate at which biochemical
reactions proceed but not altering the direction or nature of the reactions.

Coenzyme An organic, nonprotein molecule that binds with an apoenzyme (a protein

molecule) to form an active enzyme. Coenzymes are often derived from
vitamins.

Endoenzyme An enzyme which is not secreted or exported out of the cell, but is kept
and used by the cell which made it.

Substrate The specific molecule an enzyme acts upon.

Metabolism All of the organized chemical reactions in a cell which are under the
control of enzymes.

Catabolic Reactions in which chemical compounds are broken down.

reactions

Anabolic Reactions in which chemical compounds are synthesized.

reactions

Enzyme The attachment of an enzyme to a solid matrix so that it cannot escape

immobilization but can still act on its substrate.

Enzyme The disappearance of activity of an enzyme (in vivo or in vitro) due to

inactivation presence of inhibitor molecules or inhibitory conditions (changes in pH,
temperature, salt concentration etc.).

Enzyme Quantitative characteristics of enzymatic reactions.

kinetics

3
 Enzymes are synthesized within cells and, under certain conditions, can pass through
cell membranes. Enzymes that act inside the cell are called endoenzymes, while those that are
released outside the cell are known as exoenzymes. Enzymes involved in energy production
belong to the first category, whereas digestive enzymes belong to the second.

Although enzymes are basically proteins, they may sometimes have a non-protein
component attached to the protein part. This component may be an organic compound or a
metal ion. The organic compound is called a coenzyme, while the metal ion is referred to as a
cofactor. Together, the protein and non-protein parts form the holoenzyme. In some cases, the
non-protein part is so tightly bound to the protein that it cannot be separated; such a component
is called a prosthetic group. When the non-protein component dissociates from the protein part,
the enzyme loses its catalytic activity and is referred to as an apoenzyme.

Holoenzyme

Coenzymes, which are usually vitamins, sometimes become part of the active enzyme
and are indispensable for the enzyme to carry out catalysis. You have already learned about the
classification of vitamins in the previous unit and know that they are broadly classified as
water-soluble and fat-soluble. Remember that water-soluble, not fat-soluble, vitamins serve as
the precursors of coenzymes.

While participating in the catalytic mechanism, a coenzyme undergoes alteration, and

its restoration requires the involvement of another enzyme. Since coenzymes may participate
in a variety of reactions catalyzed by different enzymes, it is convenient to classify them based
on the type of element or group they transfer in these reactions—such as hydrogen, carboxyl,
amino, etc. We will learn more about the classification of enzymes in the next section.

4
 2. MECHANISM OF ENZYME ACTION
L. Michaelis and Maud L. Menten developed a general theory of enzyme action in
1913. According to their model, the enzyme (E) first binds to the substrate (S) to form a
transient enzyme-substrate complex (ES). This complex then dissociates to release the product
(P) and regenerate the unaltered enzyme (E).

Let us understand how this ES complex forms.

The action of an enzyme is initiated when the reactants i.e. substrates bind at the catalytic sites
or active sites on the enzyme molecule. The catalytic site of the enzyme molecule possesses a
complex three-dimensional form and provides a cleft, which binds the substrate as shown
herewith.

A change in the tertiary or quaternary structure of the enzyme may alter the three-dimensional
shape of the catalytic site and thus reducing its binding and catalytic activities. The ES complex
is formed mainly by non-covalent bonds between specific groups of the substrate molecules
and the specific amino acid side chains present at the catalytic site of the enzyme. Different
models for enzyme-substrate complex formation exist. Let us look at these models next.

Models for enzyme-substrate (ES) complex formation

There are two popular models to explain the enzyme-substrate interaction. These are:

• Fischer's template or lock and key model, and

• Koshland's induced fit model

The two models are described next.

Fischer 's template or lock and key model

5
According to this model, the catalytic site of the enzyme has a proper conformation compatible
to a specific substrate even in the absence of the substrate molecule, as shown in Figure 1. The
catalytic site binds the substrate and catalyzes the reaction without any change in its own three-
dimensional conformation. It has become possible to explain the specificity of many enzymes
for only one of the stereoisomers of the substrate by this model. This model, however, failed
to explain the change in enzyme activity in presence of allosteric modulators (low molecular
weight regulatory substances that bind at a specific site on the enzyme molecule other than the
catalytic site and thereby enhance or inhibit the enzyme activity).

Figure:1 Fischer's template or lock and key

model and Koshland's induced fit model

Koshland 's inducedfit model

This model considers a flexibility in the three-dimensional conformation of the catalytic site.
According to this model, despite having the required amino acids, the catalytic site of the
enzyme does not possess the conformation complementary to the substrate in absence of the
substrate molecule. Only when the substrate approaches towards the enzyme or during its
binding, the conformation of the catalytic site changes so that the enzyme can hold the substrate

6
properly, as shown in Figure 2. This model, therefore, can suitably explain the noncompetitive
inhibition and allosteric modulation of the enzyme.

Figure:2Koshland's induced fit

model
While on the topic of the mechanism of enzyme action, it is also important to know about the
unit-of enzyme activity i.e. how to express the activity' of an enzyme. Have you heard of the
term 'katal'? Recommended unit of enzyme activity •is called as 'katal', which is the amount
of an enzyme that transforms 1 mol of substrate into product in one second.

In fact, other than katal, the activity of an enzyme may be expressed in different other ways as
highlighted herewith:

a) International enzyme unit (IU) is defined as the amount of enzyme that catalyzes the
transformation of 1 g mol of substrate into product in one minute.
b) Specific activity of an enzyme preparation is expressed as kat/kg or IU/mg of protein.
c) Molar activity of an enzyme is kat/mol of the enzyme.
d) Turnover number of the enzyme is the number of molecules of substrate transformed
per catalytic site of the enzyme per minute.

3. ENZYME KINETICS

The study of the rate at which an enzyme works is called enzyme kinetics

L. Michaelis and k L. Menten developed a general theory of enzyme action and

kinetics in 1913. According to this theory, we learnt that in an enzyme substrate reaction, the
enzyme (E) first combines with the substrate (S) with the formation of an enzyme-substrate
(ES) complex, which subsequently breaks down into the product (P) and the enzyme (E) is
recovered. Thus, the enzyme catalyzed reaction proceeds in the following two steps:

(a) (b)

7
 The reactions are assumed to be reversible and K1, K2, K3 and K4 are the rate
constants of each reaction.

Let us examine enzyme kinetics as a function of the concentration of substrate

available to the enzyme. The enzyme substrate interaction may be studied with the help of
Figure 3. In this graph, substrate concentration and velocity of reaction of the enzyme catalysis
has been shown on X and Y axis, respectively. When the velocity of a reaction is plotted against
different substrate concentrations, as io the present graph, a hyperbolic curve is obtained.

Substrate Concentration
Figure:3 Effect of substrate concentration on the
reaction velocity of an enzyme catalyzed
reaction
M, N and 0 are three points on the curve representing three stages of enzyme catalyzed
reaction. While M represents that stage of the reaction when the substrate concentration is very
low and the rate of reaction is directly proportional to the substrate concentration, O represents
the stage when the reaction velocity reaches its maximum due to the gradual increase in
substrate concentration resulting in a saturation of the active site of the enzyme. In between
these two extremities lies the third point N which represents the reaction velocity of the enzyme
catalyzed reaction , that is half of the maximum velocity.

The mathematical relationship between the initial velocity of an enzyme catalyzed

reaction, the concentration of the substrate and certain characteristics of the enzyme are
expressed by the Michaelis-Menten equation, which was derived on the basis of Michaelis-
Menten theory and assumptions of G. E. Briggs and J. B. S. Haldane and was proposed in 1925.

8
The Michaelis-Menten equation is:

where, v is the velocity of the reaction at any stage,

Vmax is the maximum velocity,

[S] is the substrate concentration and

Km is the Michaelis-Menten constant, usually expressed in moles per litre.

Km represents the substrate concentration at which the velocity of the reaction is half of Vmax
Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme
and its substrate. The lower the Km the greater the affinity (so, lower is the concentration of
substrate needed to achieve a given rate).

This equation is widely used to describe the most enzyme catalyzed reactions. Now, we can
verify the situations of the three points M, N and 0 in Figure 3 with the help of this equation.

i) Point M: At this stage of enzyme-substrate reaction, the substrate concentration is

much less than Km and its value does not changes significantly with the increase in
substrate concentration. Thus, [S] can be ignored in the denominator of the
Michaelis-Menten equation. So it becomes,

As Vmax and Km are both constants, Vmax / Km ratio may be replaced by a new
constant, K. Thus, we have

So, reaction velocity (v) of the enzyme-substrate reaction at this stage is directly
proportional to substrate concentration [S].

ii. Point N: At this point, substrate concentration [S] is equal to Michaelis-Menten

constant, Km. Thus the Michaelis-Menten equation becomes:

9
 This clearly established that Km is the substrate concentration at which the reaction
velocity of an enzyme catalyzed reaction is half of maximum velocity.

3. Point O: At this point, the substrate concentration is much higher in comparison to

Km and so Km may be ignored in the Michaelis-Menten equation. Thus it becomes,
Coenzymes

Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-
Burk plot. This provides a more precise way to determine vmax and Km. Let us get to learn more
about this concept.

Lineweaver-Burk equation

Michaelis-Menten equation is often algebraically transformed to other

forms for convenience in plotting experimental data. One such form is
Lineweaver-Burk equation, which is derived by taking the reciprocal of
both sides of Michaelis-Menten equation. Thus results:

10
This is known as Lineweaver–Burk equation. Look at Figure 4. When 1/v is plotted against
1/[S], a straight line is obtained having a slope of Kₘ / Vₘₐₓ, an intercept of 1/Vₘₐₓ on the 1/v
axis and an intercept of –1/Kₘ on the 1/[S] axis.

Figure:4 Lineweaver-Burk plot

Note that Km is not a fixed value but may vary with the structure of the substrate, pH and
temperature. For enzymes having more than one substrate, each substrate has a characteristic
Km. Affinity of an enzyme for a substrate inversely varies with the Km. For example, glucose
can be acted upon by both hexokinase and glucokinase but hexokinase has lower Km value for
glucose and therefore greater affinity in comparison to glucokinase.

11
4. FACTORS AFFECTING ENZYME ACTIVITY
Rate of an enzyme substrate reaction depends on several important factors. These
include concentration of enzyme, concentration of substrate, pH, temperature etc. Without the
optimum condition of these factors, the enzymes will be unable to exhibit its best activity.
These factors are discussed briefly in this sub section.

• Concentration of enzyme
Increase in enzyme concentration will increase the rate of an enzyme catalyzed reaction. This
is because of the availability of additional catalytic sites to which the substrates can bind.
Accordingly a straight line graph is obtained as can be seen in Figure 5. Since the concentration
of substrate relative to that of enzyme is always very high, increase in enzyme concentration
results in increased enzyme activity.

Enzyme Concentration
Figure:5 Effect of enzyme concentration on enzyme
activity

• Concentration of substrate

When the concentration of the substrate is low, the rate of enzyme catalyzed reaction also
remains low, inspite of concentration of substrate being higher compared to enzyme
concentration. This is because at this stage all the catalytic sites of the enzyme are not occupied
by the substrate molecules. So, with the increase in substrate concentration, the rate of the
reaction also increases until all the catalytic sites of the enzyme are utilized. Rate of the reaction
becomes maximum at this point and beyond this, it remains constant as can be seen in Figure
6.

12
 Substrate Concentration
Figure:6 Effect of substrate concentration on
enzyme activity
• Temperature
The rise in temperature accelerates the rate of enzyme-catalyzed reaction up to a certain
temperature known as optimum temperature for the enzyme. At very high temperature, the
enzyme undergoes denaturation and subsequent loss of activity. For most enzymes, the
optimum temperatures are close to that of ambient temperature of the cell. For human beings,
the temperature is in the region of 370C while the optimum temperature of plant urease is 600C.
Certain microbial enzymes have a higher optimum temperature that enables them to adjust in
a new higher ambient temperature of a new environment.

The effect of temperature on the reaction rate is shown in Figure 7. You can see that the rate
of the reaction is almost zero at OOC and this gradually increases with the rise of temperature
until the optimum point reaches. Beyond this point, the activity of the enzyme falls due to
denaturation and the curve bends reaching ultimately the zero level. It has been found that a
rise of IOOC will double the activity of the enzyme.

Temperature

Figure:7 Effect of temperature on enzyme

activity
13
• pH
Enzymes are influenced by pH changes as they have a ionic character due to the presence of amino
and carboxylic groups. All enzymes have an optimum pH for showing highest catalytic activity
and a change of this affects their activity. However, within a narrow pH range, the changes in the
reaction rate are reversible but if the pH becomes too low or high, denaturation of the enzyme
may occur. Most enzymes have an optimum pH range between 5 and 8, although some enzymes
like pepsin and trypsin are most active at high acidic (pH 1.5) and high alkaline (pH 8) condition.
A typical curve of enzyme activity against pH changes is presented in Figure 8.

Figure:8 Effect of pH on enzyme

activity

• Activators
Activity of many enzymes is influenced by certain ions called as activators. Large number of
enzymes such as hexokinase that require ATP are also in need of divalent cations like Mg2+ or
Mn2+. Many enzymes such as ATPase require monovalent cations like Na+, K+ or NH4+ for
maximum catalytic activity. Amylase requires Cl-. Generally, these ions interact with the
substrates so that the substrates can bind with the catalytic sites of the enzyme properly. Thus, in
absence of the activators, the enzymes become inactive or sluggish.

• Oxidation
Some enzymes which have the sulfhydryl (—SH) group in the catalytic site are very sensitive
to oxidation. Due to oxidation of the —SH group by aerial oxygen or oxidizing agents, a

14
disulfide linkage (—S—S—) forms with the subsequent loss of enzyme activity. The enzyme
activity can be restored by the reduction of the enzyme by some reducing agent such as cysteine
or glutathione.

5. ENZYME INHIBITION
Enzymes are often inhibited by the presence of suitable inhibitors. Much of current drug
therapy is based on this. Basically, there are three major classes of enzyme inhibition. These
are:

• Competitive inhibition, when the substrate and inhibitor compete for binding to the
same active site

• Noncompetitive inhibition, when the inhibitor binds somewhere else on the enzyme
molecule reducing its efficiency, and

• Uncompetitive inhibition

Let us learn about each of these classes of inhibition.

• Competitive inhibition

This type of inhibition takes place when a compound having a strong structural resemblance to
the substrate competes with it for the catalytic site of the enzyme. Once the compound binds,
the enzyme cannot convert the inhibitor to products. Increasing substrate concentration,
however, is capable of displacing the inhibitor. Thus this type of inhibition is reversible in
nature. A good example of this is the reaction catalyzed by succinate dehydrogenase in the
citric acid cycle. You will learn about the citric acid cycle in Unit 6, later in this Course. In this
reaction, succinate is converted to fumarate with the aid of this enzyme. Now, the compound
malonate is

structurally similar to succinate and if present, it will compete with succinate for the catalytic
site of succinate dehydrogenase and reduce the product formation. Thus, malonate is a
competitive inhibitor for this particular reaction. The structure of succinate and malonate is
given in Figure 9.

15
 Figure:9 Structure of succinate and
malonate

In this type of inhibition, the Km of the enzyme for the substrate shows an apparent increase in
the presence of the inhibitor as can be seen in Figure 10. This means that by increasing the
substrate concentration, enzyme inhibition can be overcome. However, the Vmax remains
unaltered.

Figure:10 Lineweaver-Burk plot for an enzyme-

substrate reaction in competitive inhibition

• Noncompetitive inhibition
In this type of inhibition, the inhibitor binds at a site on the enzyme other than catalytic site.
As there is no competition between the substrate and the inhibitor, the inhibition cannot be
reversed in this case by increasing the substrate concentration. It appears that as if inhibitor is

16
removing the enzyme, thus causing a decrease in Vmax as can be seen in Figure 10. No change
of Km however, occurs.

Figure:11 Lineweaver-Burk plot for an enzyme-

substrate reaction in noncompetitive inhibition

A noncompetitive inhibitor can combine with either the free enzyme or the enzyme substrate
complex, interfering both. The most common type of noncompetitive inhibition is affected by
the substances that combine with some functional group of the enzyme (outside the catalytic
site) that is essential for maintaining the conformation of the enzyme molecule required for its
activity. For example, enzymes possessing the essential -SH group are sometimes inhibited by
metals like mercury or copper. Finally, let is get to know about the third type of enzyme
inhibition i.e. uncompetitive inhibition.

• Uncompetitive inhibition
In this type of inhibition, the inhibitor only binds with the enzyme-substrate complex making
it inactive. As a result, the product formation becomes difficult. In uncompetitive inhibition,
both Km and Vmax changes as can be seen in Figure 11. The former increases while the latter
decreases. This kind of inhibition is rare in one substrate reactions but common in two substrate
reactions.

17
 Figure:12 Lineweaver-Burk plot for an
enzyme-substrate reaction in uncompetitive
inhibition

So far we studied about the enzyme mechanism and the factors which influence and inhibit its
activity. Our study of enzymes shall be incomplete, without the understanding of the role of
enzymes and coenzymes in metabolism. The next section focuses on this aspect. But before
moving on to the next section, let us recapitulate what we learnt till now.

6. ROLE OF ENZYMES AND COENZYMES IN METABOLISM

As coenzymes participate in a variety of functions, they can be classified broadly into two
groups:

i) Hydrogen transferring coenzymes, and

ii) Group transferring coenzymes

Let's learn about these two groups of enzymes.

I) Hydrogen Transferring Coenzymes

This group consists of three important coenzymes all of which assist different enzymes in
oxidation-reduction reactions. These are:

a) Nicotinamide nucleotides
b) Flavin nucleotides
c) Lipoic acid
A brief review of these coenzymes follows.

18
(a) Nicotinamide nucleotides
These coenzymes are derived from the vitamin, niacin. You may recall reading in Unit 3 that
they are of two types, nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine
dinucleotide phosphate (NADP⁺). Collectively, they are called pyridine nucleotides. Have a
look at Figure 12. In NAD⁺, the pyridine ring is attached to a ribose molecule through
glycosidic bond and the phosphate provides a link between adenosine and nicotinamide
riboside. In NADP⁺, an additional phosphate group is present in carbon atom 2 of the ribose
molecule of adenosine component.

Oxidized and reduced forms of NAD (and NADP). The

pyridine ring of NAD⁺ is reduced by addition of a hydride
ion to C-4 when NAD⁺ is converted to NADH (and when
NADP⁺ is converted to NADPH). In NADP, 2'-hydroxyl
group of the sugar ring of adenosine is phosphorylated.

Figure:13 Oxidized and reduced forms of NAD and NADP

Both NAD+ and NADP+ act as hydrogen acceptors during oxidation-reduction

reactions in the body. Dehydrogenation is the primary mechanism of biological oxidation in

19
which two hydrogen atoms are removed from the substrate in presence of an acceptor. The
hydrogen atoms ionize to yield two H+ and two electrons.

The nicotinamide ring of NAD+ or NADP+ accepts a proton and two electrons which
are equivalent to H-. The other H+ remains as such. All reactions catalyzed by them are
reversible. Table 4.3 lists some of the enzymes, all dehydrogenases, which are dependent on
these coenzymes.

The deficiency of nicotinic acid (niacin) causes the pellagra disease. Nicotinic acid or
nicotinamide is essential as a precursor of NAD⁺ and NADP⁺ (pyridine nucleotide coenzymes)
(Figure 3b) (Sorci et al., 2010). Nicotinamide coenzymes play a role in numerous oxidation-
reduction reactions in the form of electron transfers from and to the metabolite. Pyridine ring
NAD⁺ is reduced by the addition of hydride ion onto C-4 when NAD⁺ is transformed into
NADH (and when NADP⁺ is transformed into NADPH) (Sorci et al., 2010).

NAD⁺ and NADP⁺ almost always behave as dehydrogenase substrates (Bellamacina,

1996). Dehydrogenase catalyzes the oxidation of the substrate by transferring two electrons
and proton in the form of hydride ion (H⁻) onto C-4 of nicotinamide group NAD⁺ and NADP⁺.
In this way, the reduced forms are formed (NADH and NADPH), where new C-H bond is
created on C-4 (Bellamacina, 1996).

NADH and NADPH (stable in solutions containing oxygen) possess reductive power
(Kukielka and Cederbaum, 1990). The stability of reduced pyridine nucleotides allows them to
carry their reduction potential from one enzyme to another; the characteristics not owned by
flavin coenzymes. The majority of reactions in which NADH and NADPH are formed are
catabolic reactions. The oxidation of NADH in mitochondria is coupled with ATP synthesis.
The most significant part of NADPH is used as a reduction agent in biosynthetic reactions
(Kukielka and Cederbaum, 1990).

NADH and NADPH show a maximum in ultraviolet region at 340 nm caused by

dihydropyridine ring, while NAD⁺ and NADP⁺ do not absorb the light at this wavelength. The
appearance and disappearance of the absorbance at 340 nm are useful for the measurement of
the rate of the oxidation (McComb et al., 1976).

In the mechanism of the oxidation of lactate to pyruvate catalyzed by lactate

dehydrogenase (Figure 4), the coenzyme accepts the hydride ion on C-4 in nicotinamide group.
This phenomenon leads to the bond rearrangement in the ring when electrons are moving to
20
 the positively charged nitrogen atom. The enzyme represents acid-base catalyst and the suitable
place for binding coenzyme, and the substrate as well. Two hydrogens are moving from the
lactate to produce pyruvate. One of these hydrogens are moving to NAD⁺ as a hydride ion
bearing two electrons, and the another is transferring to His-195 as a proton. The second
hydrogen is then releasing as H⁺ to regenerate the base catalyst (His-195) (Kane, 2014; Speers
and Reguera, 2012).

Enzymes requiring nicotinamide nucleotide coenzymes

Enzyme Coenzyme Substrate Product

Lactate dehydrogenase NAD⁺ Lactate Pyruvate

Alcohol dehydrogenase NAD⁺ Primary and secondary Corresponding aldehydes

alcohols and ketones

Glutamate NAD⁺ Glutamate α-ketoglutarate and

dehydrogenase ammonia

Glyceraldehyde-3- NAD⁺ Glyceraldehyde-3- 1,3-diphosphoglyceric

phosphate phosphate acid
dehydrogenase

Glucose-6-phosphate NADP⁺ Glucose-6-phosphate 6-phosphogluconolactone

dehydrogenase

Malate dehydrogenase NAD⁺ Malate Oxaloacetate

Isocitrate dehydrogenase NADP⁺ Isocitrate α-ketoglutarate

21
(b) Flavin nucleotides

Flavin nucleotides are derived from the vitamin B₂ (riboflavin) and are actively involved in
hydrogen transfer reactions. You may recall reading in the last unit about the two coenzymes
that are produced from riboflavin, i.e. flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD). Look at Figure 13 for the structure of FAD. FMN is the active component
of riboflavin and is formed by the addition of a phosphate group and FAD is formed by the
combination of FMN with one molecule of adenosine triphosphate (ATP). Both, FMN and
FAD, are involved in many oxidation-reduction reactions. Enzymes that require the presence
of FMN for their catalytic activity include glycolic acid oxidase, L-amino oxidase etc. FAD
containing enzymes are succinate dehydrogenase, D-amino oxidase etc.

22
 Figure:14 Structures of Riboflavin and its coenzyme FAD

(c) Lipoic acid

Lipoic acid acts in the transfer of hydrogen during oxidative decarboxylation reactions. It
occurs both in oxidized and reduced forms. Refer Figure 14 (a) for its structure. Figure 14 (b)
shows the structure of lipoamide, where lipoic acid is bound in an amide linkage to the ε-amino
group of a lysine residue (blue) of dehydrolipoamide acyl-transferases. The complex reactions
of the carbohydrate metabolism catalyzed by pyruvate dehydrogenase system and α-
ketoglutarate dehydrogenase system require the participation of lipoic acid.

23
 Figure:15 Structures of (a) Lipoic acid (b) Lipoamide

II) Group Transferring Coenzymes

This group consists of the following coenzymes involved in different metabolic reactions,
where the transfer of any functional group is taking place:

• Biotin

• Thiamin diphosphate (TDP)

• Pyridoxal phosphate

• Coenzyme A (CoA)

• Tetrahydrofolic acid (THF)

• Cobamide coenzymes

• Adenosine triphosphate (ATP)

• Biotin
This water soluble B group vitamin participates in the transfer of carboxylic groups. Two
important enzymes carrying out carboxylation reactions are pyruvate carboxylase and acetyl
CoA carboxylase. Pyruvate carboxylase contains four biotin molecules attached to the enzyme
protein. The biotin dependent enzymes require ATP (adenosine triphosphate) which is
converted to ADP (adenosine diphosphate) during the reaction.

24
 Figure:16 Structure of Biotin and enzyme-bound biotin

• Thaimine diphosphate
Thiamine diphosphate (TDP) is the coenzyme responsible for the transfer of aldehyde and
glyoxal groups. Figure 15 presents the structure of TDP.

Figure:17 Structure of thiamine

25 diphosphate
The most important metabolic reaction catalyzed by TDP is the oxidative decarboxylation of
α-keto acids such as pyruvic acid. The involvement of TDP in the decarboxylation of pyruvic
acid was so well known that the coenzyme was also popularly called as co-carboxylase. It is
also actively taking part in the conversion of pyruvic acid to acetyl-CoA and α-ketoglutarate to
succinyl-CoA that are catalyzed by pyruvate dehydrogenase and α-ketoglutarate
dehydrogenase complex, respectively during carbohydrate metabolism.

Another important metabolic reaction catalyzed by TDP is the conversion of xylulose-5-

phosphate to sedoheptulose-7-phosphate catalyzed by the enzyme transketolase in pentose
phosphate pathway of carbohydrate metabolism.

• Pyridoxal phosphate
Pyridoxal phosphate is derived from pyridoxine (vitamin B₆) and is involved in amino acid
metabolism. The other two compounds, pyridoxal and pyridoxamine, about which you learnt
in the last unit, having the properties of vitamin B₆ also occur as phosphate derivatives.
Enzymes that are dependent on B₆ phosphate coenzymes catalyze a variety of reactions such
as transamination (transfer of amino group from an amino acid to a keto acid), decarboxylation
(removal of carboxyl group) and racemization (transformation of one isomer to another).

26
Figure:18 Structure of pyridoxal phosphate (Pyp; Vitamin
B6)
27
Enzyme glutamate oxaloacetate transaminase catalyzes the reaction between glutamate and
oxaloacetate with the formation of a-ketoglutarate and aspartate due to transfer of one amino
group from glutamate to oxaloacetate. Pyridoxal phosphate acts as the carrier of the amino
group. Whenever it accepts the amino group, it transforms to pyridoxamine phosphate, and
after the release of the amino group, it again becomes pyridoxal phosphate. Aspartate is
decarboxylated by aspartate decarboxylase taking the help of pyridoxal phosphate.

L-alanine transforms to D-alanine by alanine racemase which also requires pyridoxal

phosphate as the co-enzyme.

• Coenzyme A
Coenzyme A is derived from the vitamin pantothenic acid. This is abbreviated as COA. This
can be divided into two components, adenosine 3,5-diphosphate and pantothenic, which is
formed by the combination of pantothenic acid and mercaptoethylamine. Refer Figure 16 to
understand its structures well. It gives rise to acyl-CoA derivatives that are mainly formed in
ATP dependent synthetase reactions. These are highly reactive and participate in various types
of reactions. For example, oxaloacetate is converted to citrate in presence of citrate synthetase
by accepting acyl group of acyl-CoA. During carbohydrate metabolism, the pyruvate is
converted by the pyruvate dehydrogenase complex to acetyl-CoA by the active participation of
coenzyme A. Also, in the oxidation of fatty acids.

Figure:19 Structure of coenzyme A

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● Tetrahydrofolate
Coenzyme tetrahydrofolate, as you may already know, is derived from the vitamin folic acid.
Have a look at the Figure 17 for structures of folic acid and tetrahydrofolate – the coenzyme.
It is responsible for the transfer of one carbon fragments at the oxidation level of formate,
formaldehyde and methanol. The two most important metabolic reactions in which
tetrahydrofolate participates are the biosynthesis of purine and methionine.

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 Figure:20 Structures of folic Acid and tetrahydrofolic acid

● Cobamide coenzymes
Cobamide coenzymes are the derivatives of vitamin B₁₂. The structure of cobamide-coenzyme
is very complex and is shown in Figure 18. They participate in many biochemical reactions. L-
methylmalonyl CoA is converted to succinyl CoA by the action of the enzyme methylmalonyl
CoA isomerase. In this reaction, the –CO–SCoA group is transferred by the cobamide-
coenzyme. An important reaction catalyzed by this coenzyme as an integral part of the
ribonucleotide reductase is the reduction of ribonucleotides to deoxyribonucleotides.

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Figure:21 Structure of vitamin Bli and its coenzyme

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● Adenosine triphosphate
Though adenosine triphosphate (ATP) often functions as a second substrate, it can also serve
as a coenzyme by modulating the activity of specific enzymes. The compound consists of
adenine connected to a ribosyl 5’-triphosphate. See Figure 19 for its structure. As a co-
substrate, ATP is utilized by various kinases for the transfer of the terminal phosphate group
to various acceptors. For example, glucose is converted to glucose 6-phosphate in presence of
the enzyme hexokinase by accepting phosphate from ATP.

Figure:22 Structure of ATP

• Lipoamide
Coenzyme lipoamide is a protein binding form of lipoic acid. Although lipoic acid is often
described as vitamin B, it seems that the animals are capable of synthesizing it. It is necessary
for particular bacteria and protozoa to grow (Figure 20) (Shen et al., 2011).

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 Figure:23 Lipoamide

It is believed for lipoamide to function as a pendulum which carries acyl groups between active
centers in multi-enzymatic complexes. For example, in complex of pyruvate dehydrogenase,
disulfide ring of lipoamide prosthetic group reacts with HETPP, binding its acetyl group on
sulfur atom attached to C-8 of lipoamide and creating thioester. Then, the acyl group is moved
to the sulfur atom of coenzyme A giving reduced (dihydrolipoamide) form of prosthetic group.

The last step catalyzed by pyruvate dehydrogenase complex is the oxidation of

dihydrolipoamide. In this reaction, NADH is formed by the action of the flavoprotein
component of the complex. The actions of multiple coenzymes of pyruvate dehydrogenase
complex show how coenzymes supplying reactive groups and thus increasing catalytic
diversity of proteins are used for storing energy and carbon building blocks (Horton et al.,
2006; Shen et al., 2011).

• Ubiquinone
Ubiquinone is a coenzyme soluble in fats synthesized by all species. In the membrane,
ubiquinone transports electrons between enzymatic complexes situated in the membrane. Some
bacteria use menaquinone instead of ubiquinone. Ubiquinone analog called plastoquinone has
a similar function in the photosynthetic transport of electrons in chloroplasts

33
.

34
 Figure:24(a) Ubiquinone; (b) Plastoquinone;
(c) Three oxidation states of ubiquinone

Ubiquinone is a stronger oxidation reagent than both NAD+ and flavin coenzymes. Similarly
to FMN and FAD, ubiquinone can accept or donate electrons (one or two) because it has three
oxidation states: oxidized Q, partially reduced semiquinone free radical and completely
reduced QH2 ..

Coenzyme Q plays the leading role in the electron transport connected to the membrane. It is
responsible for the moving of protons from one side of the membrane to another by the process
known as cycle Q. The created protein gradient leads to ATP synthesis (Lenaz et al., 2007).

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• Protein coenzymes

Some proteins behave as coenzymes. They do not catalyze reactions, but they are necessary
from specific enzymes. These enzymes are called either protein for groups transfer or protein
coenzymes. They contain functional group either as a part of their protein skeleton or as a
prosthetic group (Horton et al., 2006). Metal ions, iron-sulfur clusters, and heme groups are
reactive centers usually found in these protein coenzymes. Several protein coenzymes have
two reactive centers. Thioredoxins are observed as reduction agents (cycle of citric acid,
photosynthesis, and synthesis of deoxyribonucleotides). Disulfide reactive center of
thioredoxin is on the surface of the protein, so it is available to the active centers of
corresponded enzymes (Horton et al., 2006; Johnson et al., 2014).

• Cytochromes

Cytochromes are protein coenzymes containing heme where Fe (III) atoms undergo reversible
one-electron reduction. They are classified as a, b and c based on their visible absorption
spectra. Heme of cytochrome b type is the same as that of hemoglobin and myoglobin. Heme
of cytochrome a has a hydrophobic chain of 17 carbons on C-2 porphyrin ring and formyl group
on C-8, while heme of type b has a vinyl group attached on C-2 and methyl group on C-8. In
cytochromes of type c, the heme is covalently bound to apoprotein with two thioester bonds
formed by the addition of thiol groups of two cysteine residues for vinyl groups of the heme
(Heldt and Piechulla, 2011). The tendency to transfer an electron to another substance,
measured as a reductive potential, also varies among cytochromes. The range of reduction
potentials among prosthetic groups is an essential property of membrane-connected electron
transferred cycles and biosynthesis (Heldt and Piechulla, 2011).

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