BIOCHEMISTR

Y
ASSIGNMENT TOPICS
1st : Glycolysis
2nd : Citric Acid Cycle

By:
Student Name : Kanze-ul-Eman
Roll No : 2K20 / MZOO / 52
Semester : 1st
Program : MSC (Zoology Previous)
 GLYCOLYSIS

The series of biochemical reaction in which glucose is broken down to pyruvate

with the release of useable energy in the form of ATP Glycolysis is at the hub of
carbohydrate metabolism because virtually all sugars whether arising from the diet or
from catabolic reactions in the body can ultimately be converted to or from glucose.
Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate
supply of oxygen. This series of ten reaction is called aerobic glycolysis because oxygen
is required to reoxidize the NADH formed during the oxidation of glyceraldehyde
3-phosephate Aerobic glycolysis sets the stage for the oxidation decarboxylation of
pyruvate to acetyl CoA, a major fuel of the TCA (or citric acid) cycle.
 Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD. The
conversion of glucose to lactate is called anaerobic glycolysis because it can occur
without the participation of oxygen. Anaerobic glycolysis allows the production of ATP in
tissues that lack mitochondria (for example, red blood cell) or in cells deprived of
sufficient oxygen.

In Glycolysis, Glucose a six carbon molecule is degraded through sequential

enzyme dependent reactions into two molecules of pyruvic acid, a three carbon
compound.

Glucose is a stable molecule. It has little tendency to breakdown into simpler

products. If the energy locked in its molecular configuration is to be released, the
glucose must first be made more reactive. A small amount of energy must be invested
by the cell to initiate glycolysis. It is adenosine tri phosphate (ATP) that provides the
energy for initiating glycolysis.
 The first step in glycolysis is the transfer of phosphate group from ATP to No.6
carbon of glucose. Adenosine di phosphate and glucose 6-phosphate are formed. After
an enzyme catalyses, the conversion of glucose 6-phosphate to its isomer fructose-6-
phosphate (F-6-P). Another molecule of ATP is invested which transfers its phosphate
group this time to No.1 carbon of F-6-P forming fructose-1, 6-di phosphate and ADP.

These reactions are known as phosphorylation reactions because phosphate

groups are added to glucose and fructose molecules. The next step in glycolysis is
enzymatic splitting of fructose 1, 6-di phosphate into two fragments. Each of these two
molecules contain three carbon atoms. One is called phosphoglycer aldehyde (PGAL)
and other is Dihydroxy acetone phosphate (DHAP). These two sugar molecules are
isomers to each other and are interconvertible. This is the reaction from which glycolysis
derives its name. Normally both these molecules are converted into pyruvic acid
through subsequent enzyme controlled reactions. Since two molecules of ATP are used
this part of glycolysis is the energy investment phase. In the remaining part of glycolysis
ATP molecules are synthesized hence it is called energy yielding phase.
 REACTION OF GLYCOLYSIS

In the following reaction, an enzyme dehydrogenase and a co-enzyme

nicotinamide dinucleotide NAD+ work together. The enzyme strips off two hydrogen
atoms from PGAL. These electrons are captured by NAD+. This is a redox reaction where
PGAL is oxidized by removal of electrons and NAD is reduced by the addition of
electrons. With the loss of two hydrogen atoms PGAL is converted into phosphoglyceric
acid (PGA). Now PGA picks up phosphate group (Pi) present in cytoplasm and becomes
1-3 di phosphoglyceric acid (DPGA). In the very next step DPGA loses its phosphate
group to ADP forming ATP and 3-phosphoglyceric acid. The phosphate group attached
with carbon atom No.3 of PGA changes its position to carbon atom No.2 forming an
isomer 2-phosphoglyceric acid. With removal of water molecule 2 PGA is converted into
phospho-enol pyruvic acid (PEPA). Finally phosphate group is transferred to ADP
forming ATP and pyruvic acid. Synthesis of ATP during glycolysis is known as substrate
level phosphorylation because phosphate group is transferred directly to ADP from
another molecule.

Glycolysis is the universal energy harvesting process of life. Metabolic machinery

of glycolysis is found in all organisms from unicellular bacteria and yeasts to
multicellular bodies of plants, animals and human beings. Glycolysis occurs freely in
anaerobic environment within cytoplasm without being associated with organelle or
membrane structure. Net input and output of glycolysis can be summarized as under.
 PHOSPHORYLATION OF GLUCOSE

Phosphorylated sugar molecules do not readily penetrate cell membranes, because

there are no specific transmembrane carriers for these compounds, and because they
are too polar to diffuse through the lipid core of membranes. The irreversible
phosphorylation of glucose, therefore, effectively traps the sugar as cytosolic glucose 6-
phosphate, thus committing it to further metabolism in the cell. Mammals have several
isozymes of the enzyme hexokinase that catalyze the phosphorylation of glucose to
glucose 6-phosphate.

1- Hexokinase:
In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three
regulatory enzymes of glycolysis (see also phosphofructokinase and pyruvate kinase).
Hexokinase has broad substrate specificity and is able to phosphorylate several hexoses
in addition to glucose. Hexokinase is inhibited by the reaction product, glucose 6-
phosphate, which accumulates when further metabolism of this hexose phosphate is
reduced. Hexokinase has a low K m (and, therefore, a high affinity) for glucose. This
permits the efficient phosphorylation and subsequent metabolism of glucose even when
tissue concentrations of glucose are low. Hexokinase, however, has a low V max for
glucose and, therefore, cannot sequester (trap) cellular phosphate in the form of
phosphorylated hexoses, or phosphorylate more sugars than the cell can use.

2- Glucokinase:

In liver parenchymal cells and β cells of the pancreas, glucokinase (also called
hexokinase D, or type IV) is the predominant enzyme responsible for the
phosphorylation of glucose. In β cells, glucokinase functions as the glucose sensor,
determining the threshold for insulin secretion. In the liver, the enzyme facilitates
glucose phosphorylation during hyperglycemia. Hexokinase also serves as a glucose
sensor in neurons of the hypothalamus, playing a key role in the adrenergic response to
hypoglycemia. Despite the popular but misleading name glucokinase, the sugar
specificity of the enzyme is similar to that of other hexokinase isozymes.
 Energy Yield from Glycolysis

Despite the production of some ATP during glycolysis, the end products, pyruvate or
lactate, still contain most of the energy originally contained in glucose. The TCA cycle is
required to release that energy completely.

1. Anaerobic Glycolysis:

Two molecules of ATP are generated for each molecule of glucose converted to two
molecules of lactate. There is no net production or consumption of NADH.
2. Aerobic Glycolysis:

The direct consumption and formation of ATP is the same as in anaerobic

glycolysis — that is, a net gain of two ATP per molecule of glucose. Two
molecules of NADH are also produced per molecule of glucose. Ongoing aerobic
glycolysis requires the oxidation of most of this NADH by the electron transport
chain, producing approximately three ATP for each NADH molecule entering the
chain. NADH cannot cross the inner mitochondrial membrane, and substrate
shuttles are required.
 nd
2 Citric Acid Cycle

It is also known as TriCarboxylic Acid (TCA) cycle. In prokaryotic cells, the citric acid cycle
occurs in the cytoplasm; in eukaryotic cells, the citric acid cycle takes place in the matrix
of the mitochondria.

The cycle was first elucidated by scientist “Sir Hans Adolf Krebs" (1900 to 1981). He
shared the Nobel Prize for physiology and Medicine in 1953 with Fritz Albert Lipmann,
the father of ATP cycle.
Reaction of TCA cycle

An enzyme strips CoA from Acetyl CoA. The remaining acetyl fragment reacts
with four carbon compound oxalo acetic acid to form 6-carbon compound, citric acid.
One molecule of water is used and co-enzyme A is recycled again. Citric acid possesses
three carboxyl groups. Hence, Kreb's cycle is also known as tricarboxylic acid cycle or
(TCA Cycle).
A molecule of water is removed and another added back so that Citric acid is
isomerised to isocitric acid through Cis-aconitic acid.
Isocitric acid undergoes an oxidative decarboxylation reaction. It is first oxidized yielding
a pair of electrons (2H) that reduces a molecule of NAD to NADH+H. The reduced
carbohydrate intermediate is decarboxylated. With the removal of CO 2, molecule a 5
carbon compound α-ketoglutaric acid is formed.
α-ketoglutaric acid is again oxidatively decarboxylated. A CO 2, molecule is lost.
The remaining four carbon compound is oxidized by transfer of a pair of electron (2H +)
reducing NAD+ to NADH+H+. The four carbon fragment combines with C OA by an
unstable bond forming succiny ! C OA. Substrate level phosphorylation takes place in the
next step. CoA is replaced by phosphate group which is then transferred to Guanosine di
phosphate (GDP) to form Guanosine tri phosphate (GTP).

GTP transfers its phosphate group to ADP forming ATP. With addition of water
molecule succinic acid is formed.

With loss of two electrons (2H+) Succinic acid is converted to Fumaric acid and
FADH2, are formed. With addition of one water molecule fumaric acid is converted to
malic acid. The last step in kreb's cycle is regeneration of oxalo acetic acid. This is
formed by removal of electrons (2H+) from malic acid to NAD+ forming NADH+H+.

Glucose molecule splits into two molecules of pyruvic acid during glycolysis. Thus
two turns of cycle are required for each glucose molecule. For each pyruvic acid
molecule, three carbon atoms are removed as CO 2, and five pairs of hydrogen atoms are
used to reduce NAD and FAD to NADH+H + and FADH2, the carrier molecules. The inputs
and outputs of kreb's cycle are shown as under.

Pyruvic acid + 3H2O + 5 carriers  3CO2, + 5 carriers (2H+)

1. Component enzymes:
The pyruvate dehydrogenase complex (PDH complex) is a multimolecular aggregate of
three enzymes, pyruvate dehydrogenase (PDH or E 1, also called a decarboxylase),
dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E 3). Each catalyzes a
part of the overall reaction. Their physical association links the reactions in proper
sequence without the release of intermediates. In addition to the enzymes participating
in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound
regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase
phosphatase.

2. Coenzymes:

The PDH complex contains five coenzymes that act as carriers or oxidants for the
intermediates of the reactions. E 1 requires thiamine pyrophosphate (TPP), E 2 requires
lipoic acid and CoA, and E3 requires FAD and NAD+.

Deficiencies of thiamine or niacin can cause serious central nervous system problems.
This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the
PDH complex is inactive. Wernicke-Korsakoff, an encephalopathy psychosis syndrome
due to thiamine deficiency, may be seen with alcohol abuse.

3. Regulation of the pyruvate

dehydrogenase complex:

Covalent modification by the two regulatory enzymes that are part of the complex
alternately activate and inactivate E1 (PDH). The cyclic AMP-independent PDH kinase
phosphorylates and, thereby, inhibits E1, whereas PDH phosphatase
dephosphorylates and activates E1. The kinase itself is allosterically activated by ATP,
acetyl COA, and NADH. Therefore, in the presence of these high-energy signals, the
PDH complex is turned off. Pyruvate is a potent inhibitor of PDH kinase. Therefore, if
pyruvate concentrations are elevated, E1, will be maximally active. Calcium is a
strong activator of PDH phosphatase, stimulating E1 activity. This is particularly
important in skeletal muscle, where release of Ca2+ during contraction stimulates
the PDH complex, and thereby energy production. [Note: Although covalent
 regulation by the kinase and phosphatase is key, the complex is also subject to
product (NADH, acetyl CoA) inhibition.]

ENERGY PRODUCED BY THE TCA CYCLE

Two carbon atoms enter the cycle as acetyl CoA and leave as CO 2. The cycle does not
involve net consumption or production of oxaloacetate or of any other intermediate.
Four pairs of electrons are transferred during one turn of the cycle: three pairs of
electrons reducing three NAD+ to NADH and one pair reducing FAD to FADH 2. Oxidation
of one NADH by the electron transport chain leads to formation of approximately three
ATP, whereas oxidation of FADH2 yields approximately two ATP. The total yield of ATP
from the oxidation of one acetyl CoA. Summarizes the reactions of the TCA cycle.
REGULATION OF THE TCA CYCLE

In contrast to glycolysis, which is regulated primarily by phosphofructokinase, the TCA

cycle is controlled by the regulation of several enzyme activities. The most important of
these regulated enzymes are those that catalyze reactions with highly negative ΔGᵒ
citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase
complex. Reducing equivalents needed for oxidative phosphorylation are generated by
the pyruvate dehydrogenase complex and the TCA cycle, and both processes are
upregulated in response to a rise in ADP.

Significance of Krebs Cycle

1. Intermediate compounds formed during Krebs cycle are used for the synthesis of
biomolecules like amino acids, nucleotides, chlorophyll, cytochromes and fats etc.

2. Intermediate like succinyl CoA takes part in the formation of chlorophyll.

3. Amino Acids are formed from α-Ketoglutaric acid, pyruvic acids and oxaloacetic acid.

4. Krebs cycle (citric Acid cycle) releases plenty of energy (ATP) required for various
metabolic activities of cell.

5. By this cycle, carbon skeleton are got, which are used in process of growth and for
maintaining the cells.