Q1.
The
three major junctions of intermediary metabolism are glucose-6-phosphate,
acetyl Co-A and pyruvate.
1.
Glucose 6-phosphate
Glucose entering a cell is rapidly
phosphorylated to glucose 6-phosphate and is subsequently stored as glycogen,
degraded to pyruvate, or converted into ribose 5-phosphate. Glycogen is formed
when glucose 6-phosphate and ATP are abundant in the cell. In contrast, glucose
6-phosphate flows into the glycolytic pathway when ATP or acetyl Co-A for biosynthesis
are required. The third fate of glucose 6-phosphate is to flow through the
pentose phosphate pathway, which provides NADPH for reductive biosynthesis and
ribose 5-phosphate for the synthesis of nucleotides. Glucose 6-phosphate can be
formed by the mobilization of glycogen or it can be synthesized from pyruvate
and glucogenic amino acids by the gluconeogenic pathway.
2.
Pyruvate
This three-carbon α-ketoacid is another major
metabolic junction. Pyruvate is derived primarily from glucose 6-phosphate,
alanine, and lactate. Pyruvate can be reduced to lactate by lactate
dehydrogenase to regenerate NAD+. The lactate formed in active
tissue is subsequently oxidized back to pyruvate, in other tissues. Another
readily reversible reaction in the cytosol is the transamination of pyruvate, and
α-ketoacid, to alanine, the corresponding amino acid. Conversely, several amino
acids can be converted into pyruvate. Thus, transamination is a major link
between amino acid and carbohydrate metabolism. A third fate of pyruvate is its
carboxylation to oxaloacetate inside mitochondria, the first step in
gluconeogenesis. The carboxylation of pyruvate is also important for replenishing
intermediates of the citric acid cycle. A fourth fate of pyruvate is its
oxidative decarboxylation to acetyl CoA. This irreversible reaction inside
mitochondria is a decisive reaction in metabolism: it commits the carbon atoms
of carbohydrates and amino acids to oxidation by the citric acid cycle or to
the synthesis of lipids. Pyruvate is rapidly converted into acetyl CoA only if
ATP is needed or if two-carbon fragments are required for the synthesis of
lipids.
3.
Acetyl CoA
The major sources of this activated
two-carbon unit are the oxidative decarboxylation of pyruvate and the β-oxidation
of fatty acids. Acetyl CoA is also derived from ketogenic amino acids. The
acetyl unit can be completely oxidized to CO2 by the citric acid
cycle. Alternatively, 3-hydroxy-3-methylglutaryl CoA can be formed from three molecules
of acetyl CoA. This six-carbon unit is a precursor of cholesterol and of ketone
bodies. A third major fate of acetyl CoA
is its export to the cytosol in the form of citrate for the synthesis of fatty
acids.
Q2.
Discuss
the key regulatory steps in glycolysis and the Tricarboxylic Acid Cycle
Answer
First
regulatory step the action of hexokinase or glucokinase
This enzyme is strongly inhibited by the
product of its reaction, glucose-6-phosphate, thus preventing the unnecessary
utilization of ATP which could be used for other metabolic process. But
glucokinase the isozyme of hexokinase found in the liver and pancreas has a
higher Km and is not subject to feedback inhibition by glucose-6-phosphate.
The lower Km of hexokinase is important for tissues such as those of the brain
because it allows glucose to be phosphorylated even at concentrations lower
than the normal physiological blood/tissue levels. Glucokinase is also subject
to induction/suppression of synthesis under hormonal control. The presence of
insulin increases the amount of glucokinase through the promotion of the
transcription of the glucokinase gene and vice versa when glucagon is
predominant.
Second
regulatory step the action of phosphofructokinase-1(PFK-1)
This is the most important regulatory step
in the glycolytic pathway because it’s the first committed step in this
pathway. This enzyme is allostericaly inhibited by citrate, ATP and H+ (also
called negative effectors). Citrate an intermediate of the TCA cycle inhibits
PFK-1 when its cytosolic concentrations are high. ATP is both a substrate and
in most cases an allosteric inhibitor of this enzyme; at high concentrations it
causes a decrease in the affinity of this enzyme for fructose-6-phosphate thus
increasing the Km of the enzyme. H+ is a glycolytic end product
(lactic acid), which inhibits PFK-1 and shuts off the glycolysis to prevent
lacto-acidosis. AMP, F-2-BP and Pi are positive effectors PFK-1, a decrease in the ATP
concentration causes elevated levels of AMP. This makes it an excellent signal
of the energy status of the cell and act as a positive allosteric effector of
PFK-1 thus decreasing its Km. Fructose-2, 6-bisphosphate (F-2-BP) is
formed from F-2-B by the action of phosphofructokinase-2 which is stimulated by
Pi and inhibited by citrate. In the presence of F-2-BP, PFK-1 is activated
increasing its Km.
Third
regulatory step pyruvate kinase (PK) activity
PK is drastically inhibited by high
concentrations of ATP and reduces the apparent affinity of PK for PEP. PK is
also activated by F-1,6-P, thus the activation of PFK-1 causes a subsequent
activation of PK. Acetyl Co-A acts as an allosteric inhibitor of PK, this
occurs when there is excessive production of acetyl Co-A. A high
glucagon/insulin ratio also causes a repression of the synthesis of PK leading
to reduced glycolysis and stimulation of gluconeogenesis (coarse control)
Tricarboxylic
Acid cycle (TCA cycle)
The passage of carbons of pyruvate into and
through the TCA cycle is under control at two levels.
Ø The conversion of pyruvate to acetyl Co-A for entry into the cycle
Ø Regulation by key enzyme in the TCA under the allosteric influence
of cofactors and intermediates
Pyruvate dehydrogenase complex is regulated
by fine control mechanism
Allosteric control
The transacetylase component (E2) is
inhibited by acetyl- CoA and activated by CoASH
The dihydrolipoamide dehydrogenase
component (E3) is inhibited by NADH and activated by NAD+
ATP is an allosteric inhibitor of the
complex whiles AMP is an activator of it.
Covalent
modification
NADH and acetyl-CoA do not only inhibit the
dephospho-form (active) of the PDH complex but also activate PDH kinase leading
to the phosphorylation and the inactivation of the complex
Free CoASH and NAD+ inhibit the PDH kinase,
thus activating the PDH complex
Pyruvate is a potent inhibitor of the PDH
kinase and therefore in the presence of elevated tissue pyruvate levels, the
kinase will be inhibited and the PDH complex maximally activated.
Regulation
of the TCA cycle it self
The regulation is three exergonic steps in
the cycle catalysed by (i) citrate synthase, (ii) isocitrate dehydrogenase and
(iii) α-ketoglutarate dehydrogenase
(i)
The availability of substrate
for this enzyme (acetyl Co-A and
oxaloacetate) depends on the metabolic state of the cell and thus may limit the
rate of citrate formation
(ii)
And (iii)
NADH a product of
dehydrogenation reactions accumulates under some conditions and at high NADH/NAD+
ratio both are severely inhibited
Product
accumulation: the accumulation of the products of all the three limiting steps
inhibits feedback inhibit their respective enzymes
Respiratory control: anything that affects
the supply of O2, ADP and reducing equivalent would shut down the
cycle which is sometimes referred to as coarse control.
Q3
Discuss
the control and regulation of glycogenolysis: Indicate key enzymes and
secondary messengers that are involved in this.
ANSWER
Glycogenolysis is the catabolism of
glycogen and is catalysed by the enzyme glycogen phosphorylase. It is a
regulatory enzyme and hence subject fine control by allosteric effectors and
covalent modification and it catalyses the first step in this pathway. The
enzyme glycogen phosphorylase is allostericaly activated by AMP and
allostericaly inhibited by glucose and ATP.
Glycogen phosphorylase is also subject to
covalent modification, the enzyme exist in two forms a (the active form) and b
(the inactive form). These forms of the enzyme undergo interconvertions by the
actions of phosphorylase kinase and phosphoprotein phosphatase as described
below
Q4
Distinguish
between non-equilibrium and near equilibrium reactions in metabolic pathways
and explain the need for regulation in metabolic pathways.
ANSWER
In near equilibrium reactions the rate of
the forward reactions equal the rate of the backward reaction and there is no
net flux in either direction, such reactions are readily reversible but in
non-equilibrium reactions are those reactions that occurs in only one direction
(rate of the forward and the reverse reactions are not equal) as such there is
a net flux in one direction, such reactions are usually irreversible.
Metabolic pathways need to be controlled
and regulated for various reasons which include;
Ø The fact that organisms
feed intermittently, most organisms are not
continually feeding even though the requirement for cell energy and building
material remains fairly constant. As such there should be a mechanism to ensure
that excess fuel molecules are stored after meal released when required. Thus
the rate of utilization of fuel molecules is dependent on their supply to the
cell. For example an organism’s rate of utilization of glucose depends on several factors which include the release of
insulin/glucagon from the pancreas.
Ø The need to maintain
homeostasis, for organisms to survive they need to
maintain a constant internal environment. For example glucose needs to be
maintained at 5mM. The consumption of a carbohydrate rich diet may increase the
blood glucose to 12mM; under these circumstances every cell increases their
rate of glucose utilization to bring the level to normal. Conversely when the
level of glucose falls, it utilization is minimized to conserve it for the
brain, also the liver sets into motion processes that begins the synthesis of
glucose from non-carbohydrate sources. Thus the ability to maintain glucose
homeostasis depends on the mechanisms that alter the rate of glucose metabolism
depending on metabolic circumstances.
Ø The need to satisfy the
peculiar demands of various tissues and organs, the
brain, liver and adipose tissue differ not only in their functions but also in
their energy requirements and preferences. For example the brain utilizes
glucose as the major energy source while resting muscles prefer fatty acids.
Since the composition of food ingested may vary, it may be necessary to alter
the relative amounts of the various fuel molecules in order to satisfy the
demands of every type of cell. This is the responsibility of the liver and
involves regulation of the rates of the various metabolic pathways in the
hepatocytes.
Ø Variations in levels of
physical activity, the consumption of energy is
proportional to the intensity of physical activity. Then since organisms do not
undergo a constant level of physical activity the consumption of muscles
varies. Hence mechanisms involved in the control of energy metabolism are
closely linked to the level of physical activity.
Q5
Enumerate
four central features of metabolic pathways and explain them.
ANSWER
1.
Coordination and regulation of biosynthetic pathway; most of these pathways are not independent and thus they need to
be regulated and coordinated to ensure a certain degree of order in the system.
For example the biosynthesis of glucose from acetyl Co-A can only occur place
when there is the need for it but there is no need mobilizing resources to make
glucose when there are other glucose sources such as glycogen is available. The
glucose synthesis can only take place when tissues such as the brain which uses
glucose as it sole source of energy is glucose is in the need of it. Hence all metabolic pathways are regulated and
coordinated to ensure efficient utilization of resources.
2.
ATP is
the universal currency of energy. The high phosphoryl transfer potential of
ATP enables it to serve as the energy source in muscle contraction, active
transport, signal amplification, and biosynthesis. The hydrolysis of an ATP
molecule changes the equilibrium ratio of products to reactants in a coupled
reaction by a factor of about 108. Hence, a thermodynamically unfavourable
reaction sequence can be made highly favourable by coupling it to the
hydrolysis of a sufficient number of ATP molecules.
3.
ATP is
generated by the oxidation of fuel molecules such as glucose, fatty acids, and
amino acids. The common intermediate in most of these oxidations is acetyl
CoA. The carbon atoms of the acetyl unit are completely oxidized to CO2
by the citric acid cycle with the concomitant formation of NADH and FADH2.
These electron carriers then transfer their high potential electrons to
the respiratory chain. The subsequent flow of electrons to O2 leads
to the pumping of protons across the inner mitochondrial membrane. This proton
gradient is then used to synthesize ATP. Glycolysis also generates ATP, but the
amount formed is much smaller than that in oxidative phosphorylation. The
oxidation of glucose to pyruvate yields only 2 molecules of ATP, whereas the
complete oxidation of glucose to CO2 yields 30 molecules of ATP.
4.
NADPH is the major electron donor in reductive biosynthesis. In most biosynthesis, the products are more reduced than the
precursors, and so reductive power is needed as well as ATP. The high-potential
electrons required to drive these reactions are usually provided by NADPH. The
pentose phosphate pathway supplies much of the required NADPH.
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