Code
1.
Questions
Explain
gluconeogenesis. Add
a note on Cori cycle.
Answers
Gluconeogenesis
The ability to synthesize glucose from common metabolites is very important to most
organisms. Human metabolism, for example, consumes about 160±20 grams of
glucose per day, about 75% of this, in the brain. Body fluids carry only about 20
grams of free glucose, and glycogen stores normally can provide only about 180 to
200 grams of free glucose.
Fatty acids also act as a source for gluconeogenesis through the formation of
oxaloacetate. The term used to describe this activity is gluconeogenesis, which
means the generation (genesis) of new (neo) glucose. Synthesis of glucose from
three and four carbon precursors is essentially a reversal of glycolysis.
Gluconeogenesis Pathway
The steps involved in gluconeogenesis are as follows
*
Pyruvate carboxylase converts pyruvate to oxaloacetate in the presence of ATP
and CO2.
*
In the cytosol, phosphoenolpyruvate carboxykinase converts oxaloacetate to
phosphoenolpyruvate.
*
Phosphoenolpyruvate undergoes the reversal of glycolysis until fructose 1, 6diphosphate is produced. The enzyme fructose 1, 6- diphosphatase converts
fructose 1, 6- diphosphate to fructose 6 – phosphate.
*
Glucose 6 – phosphatase catalyses the conversion of glucose
6- phosphate to glucose.
Cori Cycle
This cycle is also called as ‘lactic acid cycle’. During strenuous exercise glycogen is
broken down to lactic acid in the muscle. Muscles lack enzyme needed to convert
pyruvate to glucose - 6-P. Therefore, it must be sent to liver (Fig.2.4). The lactate is
transferred to the liver via the blood where it is reconverted to pyruvate by pyruvate
dehydrogenase and then to glucose by gluconeogenesis. Then the glucose formed
by gluconeogenesis is taken to muscle via blood. Thus, liver and muscle are linked
by the blood stream in a metabolic cycle known as the Cori cycle named in honour of
Carl and Gerty Cori, who first described it.. The interrelationship is shown by Cori’s
cycle (Lactic acid cycle).
2.
Explain TCA cycle
with energetics.
Citric Acid Cycle
The Citric acid cycle (Tri-carboxylic acid cycle or Krebs cycle) is a cyclic metabolic
pathway in the mitochondrial matrix. In this cycle the acetyl CoA generated by the
action of Pyruvate dehydogenase complex is used as a raw material. In eight steps,
it oxidizes acetyl residues (CH3-CO-) to carbon dioxide (CO2). Transfer of the twocarbon acetyl group from acetyl-CoA to the four-carbon oxaloacetate to yield sixcarbon citrate is catalyzed by citrate synthase. A dehydration–rehydration
rearrangement of citrate yields isocitrate. Two successive decarboxylations produce
α-ketoglutarate and then succinyl-CoA, a CoA conjugate of a four-carbon unit.
Several steps later, oxaloacetate is regenerated and can combine with another twocarbon unit of acetyl-CoA. Thus, carbon enters the cycle as acetyl-CoA and exits as
CO2. In the process, metabolic energy is captured in the form of GTP, NADH, and
enzyme-bound FADH2 (symbolized as [FADH2]).
The cycle starts with the condensation of a 2-carbon unit acetyl CoA with a four
carbon unit, oxaloacetate to form a 6 -Carbon unit citrate and CoA. This reaction is
catalysed by the enzyme citrate synthase.
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The Importance of Coupled Reactions in Glycolysis
The process of glycolysis converts some, but not all, of the metabolic energy of the
glucose molecule into ATP. The free energy change for the conversion of glucose to
two molecules of is -183.6 kJ/mol:
3.
Write the reactions of
Pentose
phosphate
Pathway and write the
significance
of
Pentose
phosphate
pathway.
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This process occurs with no net oxidation or reduction.
Link Between Glycolysis and Citric Acid Cycle
Carbohydrates, most notably glucose, are processed by glycolysis into pyruvate.
Steps involved in citric acid cycle
*
Citrate is isomerized to isocitrate by the enzyme aconitase. This is achieved in a
two stage reaction of dehydration followed by hydration through the formation of
an intermediate cis aconitate (Fig. 3.4).
*
The enzyme isocitrate dehydrogenase catalyses the oxidative decarboxylation
of isocitrate to α – ketoglutarate. The formation of NADH and the liberation of
CO2 occur at this stage.
*
Conversion of α – ketoglutarate to succinyl CoA occurs through oxidative
decarboxylation, catalysed by α–ketoglutarate dehydrogenase.
*
Succinyl CoA is converted to succinate by succinate thiokinase. This reaction is
coupled with phosphorylation of GDP to GTP.
*
Succinate is oxidized by succinate dehydrogenase to fumarate. Here FAD acts
as coenzyme and it gets reduced to FADH 2
*
The enzyme fumarase catalyses the conversion of fumarate to malate with the
addition of H2O.
*
Malate is oxidized to oxaloacetate by malate dehydrogenase. The third and final
formation of NADH occurs at this stage. The oxaloacetate is regenerated, which
can combine with another molecule of acetyl CoA and continue the cycle
Energy yield during citric acid cycle
Thus when one mole of pyruvic acid is oxidized to CO 2 and H2O by Kreb’s cycle, 15
moles of ATP is produced. Since 2 of pyruvic acid molecules are obtained per
molecule of glucose entering into glycolytic reactions, 30 ATP molecules are
synthesized.
Reactions of the Pentose Phosphate Pathway
The pentose phosphate pathway consists of two phases.
1.
Oxidative phase and 2. Non –oxidative Phase.
The Oxidative Phase
+
In this phase, two molecules of NADP are reduced to NADPH, utilizing the energy
from
the
conversion
of glucose-6-phosphate into ribulose
5-phosphate
The first reaction of this phase is the oxidation of glucose 6-phosphate by glucose 6phosphate dehydrogenase (G6PD) to form 6-phosphoglucono-lactone, an
+
intramolecular ester. NADP is the electron acceptor, and the overall equilibrium lies
far in the direction of NADPH formation. The lactone is hydrolyzed to the free acid
6-phosphogluconate by a specific lactonase, then 6-phosphogluconate undergoes
oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form the
ketopentose ribulose 5-phosphate. This reaction generates a second molecule of
NADPH. Phosphopentose isomerase converts ribulose 5-phosphate to its aldose
isomer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends
at this point, and its overall equation is,
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Oxidative phase of Pentose Phosphate Pathway
Non-oxidative phase
In tissues that require primarily NADPH, the pentose phosphates produced in the
oxidative phase of the pathway are recycled into glucose 6-phosphate. In this nonoxidative phase, ribulose 5-phosphate is first epimerized to xylulose 5-phosphate:
Then, in a series of rearrangements of the carbon skeletons, six five-carbon sugar
phosphates are converted to five six-carbon sugar phosphates, completing the cycle
and
allowing
continued
oxidation
of
glucose
6-phosphate with production of NADPH. Continued recycling leads ultimately to the
conversion of glucose 6-phosphate to six CO2. Two enzymes unique to the pentose
phosphate pathway act in these interconversions of sugars: transketolase and
transaldolase.
Transketolase: This enzyme catalyzes the transfer of a two-carbon fragment from a
ketose donor to an aldose acceptor. In its first appearance in the pentose phosphate
pathway, transketolase transfers C-1 and C-2 of xylulose 5-phosphate to ribose 5phosphate, forming the seven-carbon product sedoheptulose 7-phosphate. The
remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate
Transaldolase: This enzyme catalyzes a reaction similar to the aldolase reaction of
glycolysis: a three-carbon fragment is removed from sedoheptulose 7-phosphate and
condensed
with
glyceraldehyde
3-phosphate, forming fructose 6-phosphate and the tetrose erythrose
4-phosphate. Now transketolase acts again, forming fructose 6-phosphate and
glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate .
Two molecules of glyceraldehyde 3-phosphate formed by two iterations of these
reactions
can
be
converted
to
a
molecule
of
fructose
1,
6-bisphosphate as in gluconeogenesis, and finally FBPase-1 and phosphohexose
isomerase
convert
fructose
1,6-bisphosphate
to
glucose
6-phosphate
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4.
Explain cyclic and
non-cyclic
photophosphorylation.
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Non- oxidative reactions of the pentose phosphate pathway which converts pentose
phosphates to hexose phosphates
Significance of Pentose Phosphate Pathway:
1) In red blood cells (erythrocytes), the major role of NADPH is to reduce the
disulfide form of glutathione to the sulfhydryl form. Reduced glutathione is
important for maintenance of the normal structure of eythrocytes and for
maintaining hemoglobin in the ferrous state [Fe(II)].
2) Deficiency of the oxidative pathway enzyme glucose-6-phosphate
dehydrogenase (G6PD) causes hematologic problems because the inability to
maintain reduced glutathione results in accumulation of damaging peroxides.
3) A Deficiency of Glucose 6-phosphate Dehydrogenase Confers an Evolutionary
Advantage in Some Circumstances :The incidence of the most common form of
glucose 6-phosphate dehydrogenase deficiency, characterized by a tenfold
reduction in enzymatic activity in red blood cells, is 11% among Americans of
African heritage. This high frequency suggests that the deficiency may be
advantageous under certain environmental conditions. Indeed, glucose 6phosphate dehydrogenase deficiency protects against falciparum malaria. The
parasites causing this disease require reduced glutathione and the products of
the pentose phosphate pathway for optimal growth. Thus, glucose 6-phosphate
dehydrogenase deficiency is a mechanism of protection against malaria, which
accounts for its high frequency in malaria infested regions of the world. We see
here once again the interplay of heredity and environment in the production of
disease.
Non-Cyclic Photophosphorylation
In non-cyclic photophosphorylation, the flow of electron is unidirectional, that is,
electron donated by PS II after passing through plastoquinones, cyt b6, cyt f,
plastocyanin and PS I eventually reaches ferredoxin which in turn donates it to
+
reduce NADP. The reduced NADP (NADPH + H ) is utilized for the reduction of CO 2
to carbohydrate level. The electron does not complete the cycle. It starts from PS II
and is drained off in the carbohydrates produced by CO2 reduction. So the ATP
synthesis resulting from this type of non-cyclic electron transport chain is known as
non-cyclic photophosphorylation (Fig.5.7). Water molecule is utilized as a source of
electron in this system. In the process, two molecules of ATP are formed per two
+
molecules of NADP reduced or one molecule of oxygen evolved or two molecules of
water oxidized.
+
+
2ADP + 2Pi + 2NADP + 2H2O
2ATP + 2NADPH + H + O2
Cyclic Photophosphorylation
This process involves only PS I and wavelength of light greater than 680 nm is used.
It has been referred to as cyclic photophosphorylation ( Electrons used in the cyclic
photophosphorylation do not come from water, i.e. water is not oxidized and oxygen
is not evolved in the process. Here the high-energy electron is passed by ferredoxin
to the cytochrome bf complex instead of to NADP. It then flows to plastocyanin and
+
back to the P700 of PSI. The resulting proton gradient generated from the H pump,
cytochrome bf complex, then drives ATP synthesis. During this cyclic photo-
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5.
Explain
fatty acid
synthesis and how it
is regulated?
6.
Briefly
explain
ketolysis, ketosis and
ketogenesis.
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phosphorylation, ATP is formed but no NADPH is made. Furthermore, since PS II is
not involved, no O2 is produced.
In summary, when electron transport is operating in noncyclic mode, via PS I and PS
II, the products are NADPH and ATP. In cyclic electron transport, on the other hand,
the sole product is ATP
Fatty Acid Synthesis:
Fatty acid synthesis (lipogenesis) was formerly considered to be merely the reversal
of the oxidation pathway. Though the essential chemistry of the two processes is
reversal of each other, fatty acid synthesis and degradation occur through two
separate pathways. The other major difference is the use of nucleotide co-factors.
+
+
Oxidation of fats involves the reduction of FADH and NADH . Synthesis of fats
involves the oxidation of NADPH. However, both oxidation and synthesis of fats
utilize an activated two carbon intermediate, acetyl-CoA.
There are two pathways for the synthesis of fatty acids in tissues. These are
1. De novo synthesis ( non-mitochondrial system or the cytoplasmic system of fatty
acid synthesis);
2. Mitochondrial system of fatty acid synthesis.
De novo synthesis
Fatty acids are synthesized mainly by a de novo synthetic pathway operating in the
cytoplasm, so it is referred to as extra-mitochondrial or cytoplasmic Fatty acid
synthase system. The process of fatty acid synthesis was studied by Feodor Lynen,
who won Nobel Prize in 1964. The pathway is referred as Lynen’s spiral.
Steps involved in Fatty Acid Synthesis
1. Origin of Cytoplasmic Acetyl-CoA
2. Carboxylation of acetyl-CoA to malonyl-CoA:
3.
Elongation of fatty acid
Steps involved in Synthesis of Palmitic Acid
1. Formation of acetoacyl-ACP:
2. Formation of palmitic acid from acetoacyl ACP
3. Release of palmitate from the multi-enzyme complex
Mitochondrial system of fatty acid synthesis
This system involves a series of reactions that are essentially the reverse of those by
which fatty acids are oxidized. It may be noted that all the steps of the oxidation of
fatty acids are reversible except the conversion of the α-β-unsaturated acyl-CoA, to
the corresponding beta-keto compound. An additional enzyme enoyl-CoA reductase
is used and this requires the presence of reduced NADH as its co-enzyme, which
donates hydrogen for the formation of saturated fatty acid. This system of
biosynthesis is particularly concerned with the synthesis of long chain fatty acids,
mainly stearic acid. Elongation of fatty acids occurs in both the mitochondria and
endoplasmic reticulum (microsomal membranes). Elongation involves condensation
of acyl-CoA groups with malonyl-CoA. The resultant product is two carbons longer
(CO2 is released from malonyl-CoA as in the FAS reaction), which undergoes
reduction, dehydration and reduction yielding a saturated fatty acid.
Regulation of fatty acid synthesis
The regulation of fat metabolism occurs via two distinct mechanisms viz 1.short term
regulation and 2.
long term regulation.
1. Short term regulation: ACC is the rate-limiting enzyme in fatty acid synthesis.
This enzyme is activated by citrate and inhibited by palmitoyl-CoA and other
long chain fatty acyl-CoAs. ACC activity is also affected by phosphorylation.
Glucagon stimulation activates PKA (recall from mechanism of hormone action)
which in turn results in phosphorylation of certain serine residues in ACC
leading to decreased activity of the enzyme. On the other hand, insulin
stimulation leads to phosphorylation of ACC at sites distinct from glucagon that
result in an increased ACC activity. All these forms of regulation are defined as
short term regulation
2. Long term regulation:
Control of fatty acid synthesis also occurs by alteration of enzyme synthesis and
turn-over rates. These changes are long term regulatory effects
Ketolysis: The ketone bodies or acetone bodies pass into the blood in very small
amounts under normal circumstances. The concentration of ketone bodies in the
plasma is about 1 mg per 100 ml and the average excretion is less than 125 mg.
normally, ketone bodies are carried in the blood stream to the extrahepatic tissues
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like kidney and muscles. Ketone bodies are utilized through the conversion of βhydroxybutyrate to acetoacetate and of acetoacetate to acetoacetyl-CoA. The
acetoacetyl- CoA is then cleaved by thiolase into two molecules of acetyl-CoA. This
process of oxidation of ketone bodies is called as ketolysis.
Ketosis : Whenever the rate of ketogenesis in the liver exceeds the rate of ketolysis
in the periphery, the ketone bodies in the blood increase. This condition is called
ketonemia. At this stage, ketone bodies are excreated in detectable quantities in the
urine. This condition is called ketonuria. When ketonemia and ketonuria are marked,
the acetone which has a significant vapour pressure escapes in the exhaled air
giving rise to acetone, odour in the breath. The triad of ketonemia, ketonuria and
acetone odour in the breath is referred as ketosis. If ketosis is very severe then,
acidosis will set in, since ketone bodies need a large amount of water for their
excretion. At this stage, the person with ketosis become acidotic and dehydrated and
may even pass into coma stage.
Two main causes of ketosis are 1) starvation and 2) diabetic mellitus. In starvation
there is deprivation of carbohydrates and in diabetes carbohydrates are not
efficiently utilized. The persons survive on the store of glycogen in the liver for
energy. When this is depleted, energy is derived by the break down of fats in the
body. The accelerated fat metabolism leads to the formation of large amounts of
acetyl-CoA and ketone bodies, giving rise to ketosis and ketonuria. Large amount of
ketone bodies may also be excreta in urine due to high fat diet.
Clinical Significance of Ketogenesis: Normal physiological responses to
carbohydrate shortages cause the liver to increase the production of ketone bodies
from the acetyl-CoA generated from fatty acid oxidation. This allows the heart and
skeletal muscles primarily to use ketone bodies for energy, thereby preserving the
limited glucose for use by the brain. The most significant disruption in the level of
ketosis, leading to profound clinical manifestations, occurs in untreated insulindependent diabetes mellitus. This physiological state, diabetic keto-acidosis (DKA),
results from a reduced supply of glucose (due to a significant decline in circulating
insulin) and a concomitant increase in fatty acid oxidation (due to a concomitant
increase in circulating glucagon). Ketone bodies are relatively strong acids and their
increase lowers the pH of the blood. This acidification of the blood is dangerous
chiefly because it impairs the ability of hemoglobin to bind oxygen
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