NAME : MAKOTOSE TANAKA O (N02223087M)

: NCUBE USHER (N02223085G)

LEVEL : 1.1

PROGRAM : MBBS

COURSE : BIOCHEMISTRY

COURSE CODE : MBM 1001

LECTURER : Ms Maraura

QUESTION : Assignment 1

1. Fluoroacetate is an inhibitor of the citric acid cycle.

a) Describe the mechanism by which the metabolic poison fluoro-acetate in inhibiting

the TCA cycle

Fluoroacetate inhibits step one of the TCA cycle which is catalysed by the enzyme aconitase.
Aconitase is required for the isomerization of citrate to isocitrate. Flouroacetate is
converted to fluoro- Acetyl CoA by the enzyme acetate thiokinase. The fluoro – Acetyl CoA
produced is then converted to fluorocitrate by citrate synthase. Fluorocitrate is a potent
inhibitor of the enzyme aconitase thus inhibiting the whole TCA cycle.

b) State the sources of fluoroacetate (3 marks)

• Bigbobiaceae family of plants

• Dichapetalum cymosum

• Fabaceae family of plants

2. Discuss the regulation of blood glucose by hormones. (13 marks)

The level of blood glucose is maintained within a range of 3.1 to 6.9 mmol/dl. Thus when
blood glucose level increases above 6.9mmol/dl the hormone insulin is produced by the
beta cells of islets of Langerhans of pancreas. The action of insulin is to lower blood glucose
level. The hormone insulin will increase the uptake of glucose by the tissue cells and will
favour glycogenesis which is the synthesis of glycogen from glucose. Amylin is also secreted
into the body along with insulin and it inhibits glucagon, thus lowering blood glucose level.
Amylin also increases brain satiety in order to encourage feeling full after a meal, thereby
discouraging rapid blood glucose spikes. In addition, incretins also stimulate secretion of
insulin and reduce gastric secretions to lower high glucose levels. The hormones amylin and
insulin will inhibit the metabolic pathways such gluconeogenesis and glycogenolysis which
release glucose into blood. When blood glucose levels fall below 3.1 mmol/dl there is
stimulation of gluconeogenesis and glycogenolysis. The hormone glucagon is released by the
alpha cells of the pancreas will stimulate glycogenolysis by a signal transduction pathway
that phosphorylates glycogen phosphorylase enzyme which will break down glycogen to
glucose to elevate blood glucose level. The enzyme glucagon also acts to stimulate the
production of the enzymes for gluconeogenesis thus promoting the synthesis of glucose.
Epinephrine is hormone produced by the adrenal cortex usually by stimulation of the
sympathetic nervous system. It acts on the liver and muscle cells to increase glycogen
phosphorylase activity for increasing glycogenolysis. The hormone thyroxine produced by
the thyroid glands will also stimulate gluconeogenesis and glycogenolysis in the liver,
thereby increasing amount of glucose in the body. Glucocorticoids are hormones which are
released by the adrenal cortex. These hormones will stimulate the metabolism of proteins
and also gluconeogenesis by increasing the production of the enzymes glucose- 6-
phosphatase and fructose -1, 6 – bisphosphatase (Satyanarayan and Chakrapani, 2015).
Growth hormone and adrenocorticotropic hormone are hyperglycaemic hormones that
produced by the adrenal cortex that will increase blood glucose level. Growth hormone will
decrease the uptake of glucose by tissues while adrenocorticotropic will decrease glucose
utilization by the cells.

3. Describe the fate of pyruvate under anaerobic and aerobic conditions (5)

Under aerobic conditions pyruvate is oxidatively decarboxylated to yield acetyl CoA in a

reaction catalysed by pyruvate dehydrogenase. Acetyl CoA will then participate in the TCA
cycle.
Under anaerobic condition pyruvate will be converted to lactic acid by the enzyme Lactate
dehydrogenase. Lactate dehydrogenase is a NADH depended enzyme that uses NADH from
the glycolytic pathway and this happens in the muscles. In yeasts, an alcoholic fermentation
takes place and the pyruvate is converted to acetaldehyde by the enzyme pyruvate
carboxylase and CO2 is released. The acetaldehyde is then converted to ethanol by alcohol
dehydrogenase enzyme.

4. Give an account of the role of insulin in metabolism highlighting its biochemical and
physiological effects as well as its functions. (12 marks)

Insulin is a hormone produced by the beta cells of Langerhans in the pancreas. This
hormone has direct effect on the metabolism of carbohydrates, lipids and proteins. Insulin is
an anabolic hormone and predominantly acting as an anticatabolic hormone. Half of the
carbohydrates that are ingested are utilized in energy generation while the other half is
converted into lipids or glycogen (Satyanarayana and Chakrapani, 2015). The production of
lipids and glycogen are regulated by insulin which acts as a hypoglycaemic hormone, tends
to lower blood glucose levels when it is high. The hormone insulin has an effect of increasing
the absorption glucose by muscle and adipose tissue. The insulin has no effect on the
absorption of glucose by the liver hepatocytes but has an effect on its utilization thus insulin
will indirectly affect the uptake of glucose in liver. Insulin has an effect on the utilization of
glucose in the body and it does so by increasing the glycolytic pathway. Insulin will stimulate
key enzymes such as glucokinase, phosphofructokinase and pyruvate kinase of the glycolytic
pathway.

Insulin will have an effect that reduces glucose synthesis by reducing gluconeogenesis and
glycogenolysis. Insulin will hydrolyze cyclic AMP into 5 AMP thus preventing the activation
of the enzyme glycogen phosphorylase. Insulin will suppress gluconeogenesis by
suppressing the enzyme pyruvate carboxylase, phosphoenolpyruvate carboxykinase. In lipid
metabolism insulin has net effects of reducing free fatty acids from the fat stores in the body
to produce ketone bodies. Insulin also increases lipogenesis by stimulating acetyl CoA
carboxylase which is a key enzyme in fatty acid biosynthesis from acetyl CoA. Insulin
promotes the synthesis of triglycerides from glyceraldehyde phosphate from glycolysis and
NADP from HMP shunt. Insulin will have an effect to reduce the breakdown of lipids in
adipocytes or in the liver. Insulins acts to reduce the activity of the hormone sensitive lipase
thus the overall effect is to reduce break down of fats. Insulin reduces ketogenesis by
decreasing the activity of HMG CoA synthetase. Furthermore, insulin promotes the use of
acetyl CoA in the Krebs cycle thereby reducing its availability for ketogenesis.

In protein metabolism, since insulin is an anabolic hormone it will favour the entry of amino
acids inside the cell and decrease the breakdown of proteins. Besides the metabolic effects
of insulin, insulin promotes cell growth and replication. This is mediated through certain
factors such as epidermal growth factor, platelet derived growth factor and prostaglandins.

5. Discuss the metabolic significance of the pentose phosphate pathway and describe
the oxidative phase of the pathway. (13marks)

The glucose upon entering the cell is converted to Glucose- 6- phosphate by the enzyme
glucokinase. The glucokinase is then converted to glucose 1 phosphate by the enzyme
phosphoglucose isomerase and now the glucose is activated to enter the oxidative phase of
the HMP pathway. The first step of the pathway is the oxidative dehydrogenation of
glucose- 1- phosphate to 6 phospho glucono lactone which is a NADP dependent enzyme.
This reaction will generate NADPH.

Step two of the pathway is the hydrolysis of 6 phospho glucono lactone to 6 phospho
gluconic acid. This reaction involves addition of water and is catalysed by the enzymes
gluconolactone hydrolase. Step three of the pathway is another oxidative reaction and it
involves the oxidative decarboxylation of 6- phosphogluconic acid to ribulose- 5- phosphate
by the enzyme 6- phosphogluconate dehydrogenase. First there is dehydrogenation 6-
phosphogluconic acid to 3 keto- 6-phosphogluconate which is unstable and spontaneously
breaks down to ribulose- 5- phosphate and CO2. This reaction also yields NADPH.

The NADPH generated in the oxidative pathway is required for generating reducing
equivalents in fat acid biosynthesis. Thus NADPH is used in synthesis of steroid hormones.
NADPH is also used in the preventing of cataract formation in the lenses of the eyes by
preventing opacification. NADPH of the oxidative phase is also used in converting
methemohemoglobin which contains iron in the ferrous form into haemoglobin with iron in
the ferric state. Haemoglobin is one which carries the oxygen binding around the body.
NADPH is also involved in bactericidal action of phagocytes. NADPH oxygenase enzyme
converts molecular oxygen to superoxide which is a reactive oxygen species that kills
bacterial. It is also required by the enzyme cytochrome P450 in removal of toxins from the
body. The NADPH produces in red blood cell prevents the peroxidation of the PUF in the
membrane of red blood cells. The glutathione is oxidised to G-S-S-G during its process of
removing the reactive oxygen species (hydrogen peroxide) from the cell and is reduced back
to G-S-H in the presence of NADPH.

6. Clearly outline the digestion of carbohydrates, highlighting the related

malabsorption syndromes. (25)

The digestion of carbohydrates occurs briefly in the mouth where there is an action of
salivary alpha- amylase. After ingesting carbohydrate rich food such as sadza that has a high
content of the polysaccharide starch, the food is broken down into monosaccharaides which
are the simplest form carbohydrate and can be absorbed by the intestinal mucosa. In the
mouth the food is chewed by the teeth and broken down into small particles to increase
surface area for enzyme action. The tongue in the mouth also roles the food into a bolus
mixing it with salivary amylase. In the mouth the enzyme salivary alpha-amylase (ptyalin)
starts breaking down the 1, 4- glycosidic bonds randomly. The enzyme ptyalin is activated by
Chloride ions and it works at an optimum pH of 6,7. The end result of this brief digestion is
the alpha limit dextrin, small amounts of maltose and isomaltose. The products of this
digestion are then passed down the oesophagus by means of peristalsis and the food
reaches the stomach. Alpha amylase activity then stops in the stomach because it does not
work under acidic conditions in the stomach. However, hydrochloric acid in the stomach
helps to hydrolyse sucrose to glucose and fructose. The walls of the stomach contract to mix
the food into forming chyme which is passed into the duodenum.

In the duodenum, digestion continues with the release of bicarbonate by the pancreas
which neutralises acidic contents from the stomach. The pancreatic alpha- amylase will
break down the alpha 1, 4 glycosidic bonds in the starch into oligosaccharides, isomaltose
and maltose. Then digestion will continue in the ileum by the brush boarder enzymes on the
intestinal mucosa. The brush boarder enzyme maltase will break down maltose into glucose
by breaking down the 1, 4 glycosidic bonds. The various disaccharides from food are
digested by brush boarder enzyme. The brush boarder enzyme lactase will break down will
lactose to galactose and glucose. The sucrose enzyme, sucrose-isomaltase exists as a
complex cleaving sucrose to glucose and fructose and isomaltose. The end result of this
digestion is free glucose units. The absorption of glucose and galactose is by secondary
active transport by sodium depended glucose transport. Fructose is carried by facilitated
diffusion by GLUT5. In the process of digestion deficiance of the enzyme will result in related
malabsorption. Disaccharides are not absorbed by the intestinal mucosa thus if they are not
broken down they will result in malabsorption syndromes. Congenital deficiency of the
enzyme lactase will result in lactose intolerance where by lactose is accumulating in the
intestinal mucosa. Congenital deficiency of sucrase will result in sucrose intolerance. The
accumulation sucrose and lactose will be acted on by bacteria to form hydrogen, carbon
dioxide and methane. These are osmotically active and will result in water retention and this
results or lead to a bloated stomach (Satyanarayan and Chakrapani, 2015) . Malabsorption
syndromes will result in stomach cramps and flatulency.

7. Give an account of the chemiosmotic coupling theory which explains

energy generation. According to this theory, what are the effects of

uncouplers? (25)

Peter Mitchell proposed the chemiosmotic hypothesis in 1961. The Chemiosmotic principle
proposes that the energy released from transfer of electrons is conserved by pumping
hydrogen ions into the inter membrane space from mitochondrial matrix thus creating a
hydrogen ion electro chemical gradient across the membrane. This energy conserved in
electrochemical gradient is used to phosphorylate an ADP molecule into ATP which is the
energy currency of the cell. The electrochemical gradient contains two aspects; the chemical
gradient which is due to an increase in concentration of hydrogen ions across the
membrane and the electrical gradient due to an increase of positive charges outside the
mitochondrial matrix. It was also observed that increasing the acidity of the cytosol would in
turn increase the rate of oxidative phosphorylation. For protons to be pumped across the
membrane there must an alternation between electron carriers and hydrogen and electron
carriers in the electron transport. Complex 1, Complex 3 and Complex 4 are capable of
transporting electrons across the membrane. The proton electrochemical gradient that is
produced is coupled to the generation of ATP by the enzyme complex ATP synthase. The
free energy from the proton electrochemical gradient is dissipated by movement of
electrons back into the mitochondrial matrix via the Complex V of the electron transport
system in a process that will generate ATP. Complex V of the electron transport undergoes
rotation that is depending on proton gradient. The rotation of the beta subunit of the F1 of
Complex 5 will join together an ADP and ATP. For the chemiosmotic principle to hold there
are various factors that should be considered. The membrane should be intact and
impermeable to potassium, hydrogen, hydroxide and chloride ions whose diffusion across
the inner membrane would discharge the electrochemical gradient. As a result compounds
that reduce the permeability of the inner membrane of the mitochondria will dissipate this
electrochemical gradient without generation of energy (without it being couples to ATP
synthesis). Uncouplers are lipid soluble substances that can cross the mitochondrial inner
membrane. An uncoupler in the intermembrane space will become protonated and then it
will cross the membrane to release the proton in the mitochondrial matrix without
generation of energy. Such uncouplers include thermogenin and 2,4 dinitrophenol. This will
result in heat generation without energy production thus they are used by hibernating
animals to generate heat (Voet and Voet, 2004).

References

Satyanarayana, U. and Chakrapani, U., 2015. Biochemistry (with clinical concepts & case
studies). New Delhi: Elsevier Health Sciences APAC.

Champe, P., Harvey, R. and Ferrier, D., 2008. Lippincott's illustrated reviews. Philadelphia,
Pa. etc.: Lippincott Williams and Wilkins.

Voet, D. and Voet, J., 2004. Biochemistry. Hoboken, NJ: J. Wiley & Sons.

Chatterjea, M.N. and Shinde, R (2002) Text book of medical biochemistry. 5th Edition,
Jaypee Brothers, New Delhi.