METABOLISM BIOCHEMISTRY

1. LESSON 6&7
Overview of Carbohydrate Metabolism
& Associated Disorders

3. Regulation of Phosphofructokinase
Regulation of glycolysis.

4. Significance of glycolysis
 Glycolysis meets energy requirements of all kinds of cells.
 Anaerobic glycolysis mainly supplies energy to rapidly
contracting skeletal muscle.
 Dietary fructose and a lactose are also metabolized by this path
way.
 Glycolysis also supplies precursors for other pathways.
 For example, pyruvate is used for alanine formation and
dihydroxyacetone is used for triglyceride formation.
 In erythrocytes deficiency of pyruvate kinase causes
hemolytic anaemia.
HEALTH BYTE: Four different vitamins required in human
nutrition are vital components of this system: thiamine (in
TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate
(in CoA). We have already stated the roles of FAD and NAD as
electron carriers.
 Consequently, mutations in the genes for the subunits of the PDH
complex, or a dietary thiamine deficiency, can have severe
consequences. Thiamine-deficient animals are unable to oxidize
pyruvate normally. This is of particular importance to the
brain, which usually obtains all its energy from the aerobic
oxidation of glucose in a pathway that necessarily includes the
oxidation of pyruvate.
 People who habitually consume large amounts of alcohol can
also develop thiamine deficiency, because much of their dietary
intake consists of the vitamin-free “empty calories” of distilled
spirits. An elevated level of pyruvate in the blood is often an
indicator of defects in pyruvate oxidation due to one of these
causes.

5. 2, 3-bis phosphoglycerate cycle. or Rapoport-Leubering cycle

6.  Persons who live at high altitude undergo state of low O2 affinity for HB
due to simultaneous increase of 2,3 bisphosphoglycerate. This increase
can be reversed on returning to sea level.
 Fetal HB has less 2,3 bisphosphoglycerate than adult HB, so fetal HB
has high O2 affinity.
 During storage of blood in blood banks, there is decrease in 2,3
bisphosphoglycerate so, stored blood has high O2 affinity, which is not
suitable for blood transfusion especially to ill patients. If 2,3
bisphosphoglycerate is added to stored blood, it can’t penetrate RBCs wall.
So, it is advisable to add insoine, which is a substance that can penetrate
RBCs wall and change it into 2,3 bisphosphoglycerate through HMP
shunt.

7. Gluconeogenesis:
Significance of gluconeogenesis
 During fasting and starvation gluconeogenesis meets body glucose
requirement.
 Gluconeogenesis is the only source of glucose to organs like brain, skeletal
Muscle, erythrocytes etc. If gluconeogenesis is blocked brain dysfunction
occurs.
 Gluconeogenesis clears metabolic waste product like lactate. Excess amino
acids of dietary origin is converted to glucose by gluconeogenesis.

9. Paramedics bring a patient to the emergency department because he was found
unconscious in an alley by passers by. The man was unshaven and dishevelled, and
appeared to be about 40 years old. Blood alcohol levels were found to be 0.25% and
blood glucose levels 32 mg/dL. IV glucose was initiated, and this enabled the man to
regain consciousness, although he was still inebriated. While conscious, a history
revealed that the man was a chronic alcoholic, and as far as he could remember, he
had been only drinking for the past 2 weeks, with nothing to eat. Analysis of liver
enzyme levels in his blood revealed normal readings. Assuming that his liver is still
functioning normally, why is this patient hypoglycemic?
(A) Liver glycogen stores were depleted by the high NAD+/NADH ratio
(B) Liver glycogen stores were depleted by the high NADH/NAD+ ratio
(C) The high NAD+/NADH ratio impaired gluconeogenesis
(D) The high NADH/NAD+ ratio impaired gluconeogenesis
(E) The high NAD+ /NADH ratio impaired glycolysis The answer is D: The high NADH/NAD+ ratio impaired gluconeogenesis. Ethanol oxidation to
acetic acid (via acetaldehyde) generates large amounts of NADH. As liver glycogen stores have been
depleted within 36 h of the fast, gluconeogenesis is required to maintain blood glucose levels. The major
precursors for gluconeogenesis are glycerol, lactate, and amino acids (which give rise to pyruvate or TCA
cycle precursors, which generate oxaloacetate). Because of the high NADH/NAD+ ratio (due to the
ethanol metabolism), pyruvate destined for gluconeogenesis is shunted to lactate in order to
regenerate NAD+ to allow alcohol metabolism to continue. Similarly, oxaloacetate is shunted to
malate, also to regenerate NAD+ for ethanol metabolism. Glycerol, which is converted to glycerol-3-
phosphate, cannot go to dihydroxyacetone phosphate due to the high NADH levels in the liver. Thus, the
high NADH/NAD+ ratio diverts gluconeogenic precursors from entering gluconeogenesis, and the
liver has trouble maintaining adequate blood glucose levels. Liver glycogen stores have been depleted
within the first 36 h of the fast, but glycogen regulation is not affected by the NADH/NAD+ ratio. Under
conditions in which the liver is exporting glucose (glucagon administration, for example), liver glycolysis is
inhibited by covalent modification of key regulatory enzymes, not the NADH/NAD+ ratio. These pathways
are indicated in the figure above.
CASE STUDY:

10. Alcohol-related hypoglycemia is due to hepatic glycogen depletion combined
with alcohol-mediated inhibition of gluconeogenesis. It is very common in
malnourished alcohol abusers but can occur in anyone who is unable to ingest
food after an acute alcoholic episode followed by gastritis and vomiting/
The primary pathway for alcohol metabolism involves alcohol dehydrogenase
(ADH), a cytosolic enzyme that catalyzes the conversion of alcohol to
acetaldehyde. This enzyme is located mainly in the liver, but small amounts are
found in other organs such as the brain and stomach.
During conversion of ethanol by ADH to acetaldehyde, hydrogen ion is
transferred from alcohol to the cofactor nicotinamide adenine dinucleotide
(NAD+) to form NADH.
i. The NADH produced in the cytosol by ADH must be reduced back to NAD+ via either the
malate aspartate shuttle or the glycerol-phosphate shuttle. Thus, the ability of an individual
to metabolize ethanol is dependent upon the capacity of hepatocytes to carry out either of
these 2 shuttles, which in turn is affected by the rate of the TCA cycle in the mitochondria
whose rate of function is being impacted by the NADH produced by the ALDH reaction.
ii. The reduction in NAD+ impairs the flux of glucose through glycolysis at the
glyceraldehyde-3-phosphate dehydrogenase reaction, thereby limiting energy production.
iii. Additionally, there is an increased rate of hepatic lactate production due to the effect of
increased NADH on direction of the hepatic LDH reaction. This reversal of the LDH
reaction in hepatocytes diverts pyruvate from gluconeogenesis leading to a reduction in
the capacity of the liver to deliver glucose to the blood.
iv. Similar to lactate formation, malate is also produced from oxaloacetate. Deficiency of
oxaloacetate negatively affects gluconeogenesis as well as the functioning of TCA cycle.
v. In addition to the negative effects of the altered NADH/NAD+ ratio on hepatic
gluconeogenesis, fatty acid oxidation is also reduced as this process requires NAD+ as a
cofactor.
vi. In fact, the opposite is true, fatty acids synthesis is increased and there is an increase in
TAG production in the liver. In the mitochondria, the production of acetate from
acetaldehyde leads to increased levels of acetyl-CoA. Since the increased generation of
NADH also reduces the activity of the TCA cycle, the acetyl-CoA is diverted to fatty acid
synthesis.
vii. The reduction in cytosolic NAD+ leads to reduced activity of glycerol-3-phosphate
dehydrogenase (in the glycerol 3-phosphate to DHAP direction) resulting in increased
levels of glycerol 3-phosphate which is the backbone for the synthesis of the TAG. Both of
these two events lead to fatty acid deposition in the liver leading to fatty liver syndrome.
viii. Increased [lactate/pyruvate] ratio, results in hyperlacticacidemia. Lactate accumulation
causes lactic acidosis (metabolic acidosis)
ix. Lactate competes with uric acid for excretion, decreasing its excretion and thus
aggravating gout. Gout is a common finding in chronic alcoholics.
Much of the acetaldehyde formed from alcohol is oxidized in the liver in a
reaction catalyzed by mitochondrial NAD-dependent aldehyde dehydrogenase
(ALDH)
The product of this reaction is acetate, which can be further metabolized to CO2
and water, or used to form acetyl-CoA. As a net result, alcohol oxidation
generates an excess of reducing equivalents in the liver, chiefly as NADH. The
excess NADH production appears to contribute to the metabolic disorders that
accompany chronic alcoholism.

11. Mitochondria Shuttle System
a. Glycerol 3-phosphate Shuttle
 Skeletal muscle and brain use a different NADH shuttle,
the glycerol 3-phosphate shuttle
 It differs from the malate-aspartate shuttle in that it delivers the
reducing equivalents from NADH to ubiquinone and thus into
Complex III, not Complex I, providing only enough energy to
synthesize 1.5 ATP molecules per pair of electrons.
 Shuttle systems indirectly convey cytosolic NADH
into mitochondria for oxidation.

12. b. Malate-Aspartate Shuttle
The most active NADH shuttle, which functions in liver,
kidney, and heart mitochondria, is the malate-aspartate
shuttle
 The reducing equivalents of cytosolic NADH are first
transferred to cytosolic oxaloacetate to yield malate,
catalyzed by cytosolic malate dehydrogenase.
 The malate thus formed passes through the inner membrane
via the malate–a-ketoglutarate transporter. Within the
matrix the reducing equivalents are passed to NAD+ by the
action of matrix malate dehydrogenase, forming NADH; this
NADH can pass electrons directly to the respiratory chain.
 About 2.5 molecules of ATP are generated as this pair of
electrons passes to O2.

13. Significance of TCA Cycle:
 It is final common metabolic pathway for
oxidation of carbohydrates, lipids and
proteins.
 Intermediates of TCA cycle are used for
anabolic reactions.
 Fatty acids, cholesterol, amino acids and
porphyrins are compounds formed from
citric acid cycle intermediates.

14. Regulation of the Citric Acid Cycle
HEALTH BYTE: Some mutations in enzymes of the citric acid cycle may lead to
cancer. When the mechanisms for regulating a pathway such as the citric acid cycle are
overwhelmed by a major metabolic perturbation, the result can be serious disease.
Genetic defects in the fumarase gene lead to tumors of smooth muscle (leiomas)
and kidney; mutations in succinate dehydrogenase leads to tumors of the adrenal gland
(pheochromocytomas). In cultured cells with these mutations, fumarate (in the case of fumarase
mutations) and, to a lesser extent, succinate (in the case of succinate dehydrogenase mutations)
accumulate, and this accumulation induces the hypoxia- inducible transcription factor HIF-1a. The
mechanism of tumor formation may be the production of a pseudohypoxic state. In cells with these
mutations, there is an up-regulation of genes normally regulated by HIF-1a. These effects of
mutations in the fumarase and succinate dehydrogenase genes define them as tumor suppressor
genes.

15. Electron-Transport Chain (ETC)
(Oxidative Phosphorylation)
a) Complex I: NADH to Ubiquinone
Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase
b) Complex II: Succinate to Ubiquinone
Complex II, also called succinate dehydrogenase
c) Complex III: Ubiquinone to Cytochrome c
Complex III, also called cytochrome bc1 complex or ubiquinone: cytochrome c
oxidoreductase
d) Complex IV: Cytochrome c to O2
Complex IV, also called cytochrome oxidase
Leakage of electrons at complex I and complex III from electron transport
chains leads to partial reduction of oxygen to form superoxide. Subsequently,
superoxide is quickly dismutated to hydrogen peroxide by two dismutases
including superoxide dismutase 2 (SOD2) in mitochondrial matrix and
superoxide dismutase 1 (SOD1) in mitochondrial intermembrane space.
Collectively, both superoxide and hydrogen peroxide generated in this process
are considered as mitochondrial ROS.
Mitochondrial ROS (mtROS or mROS) are reactive oxygen
species (ROS)
HEALTH BYTE:

16. Significance of HMP Pathway:
1. NADPH produced is used for the biosynthesis of fatty acids,
cholesterol. deoxyribonucleotides, bile acids, glutamate, hormones
and detoxification by cytochrome P hydroxylase 450
2. In erythrocytes NADPH is used for removal of hydrogen
peroxide by glutathione and conversion of methemoglobin to
normal hemoglobin.
3. In neutrophils NADPH is used for superoxide biosynthesis.
4. Pentose phosphates are used for nucleic acid and nucleotide
biosynthesis.
5. Pentose of nucleic acid breakdown are used for energy production
after they are converted to intermediates of glycolysis by this
pathway.
6. Xylulose of uronic acid pathway is either converted to glucose or
intermediates of this pathway.

17. HEALTH BYTE 1: Some persons have a genetic defect in this enzyme, typically yielding
an unstable enzyme, that has shorter half-life in the RBC, or an enzyme that is unusually
sensitive to inhibition by NADPH. In either case, insufficient flux of the HMP-shunt and
so decreased production of NADPH results, and the cell’s ability to recycle GSSG to
GSH is impaired. Drug-induced oxidative stress leads to lysis of RBCs (haemolysis);
haemolytic anaemia is the obvious consequence
Wernicke-Korsakoff Syndrome
Thiamine deficiency in alcoholics does not cause beriberi but Wernicke-Korsakoff
syndrome. Reduced activity of the thiamine-dependent transketolase enzyme
occurs in this condition resulting is impairment of HMP shunt. This condition is
diagnosed by clinical features that include mental derangements, delirium and
motor incoordination, which may progress to chronic stage (called Korsakoff psychosis)
characterized by amnestic syndrome. Early diagnosis and immediate treatment is
essential, because brain damage in this condition is irreversible.
HEALTH BYTE 2:

18. FEEDER PATHWAYS FOR MONOSACCHARIDES
1. Galactose Metabolism
HEALTH BYTE 1: Lactose intolerance, common among adults of most human populations except those
originating in Northern Europe and some parts of Africa, is due to the disappearance after childhood of most or
all of the lactase activity of the intestinal epithelial cells. Without intestinal lactase, lactose cannot be completely
digested and absorbed in the small intestine, and it passes into the large intestine, where bacteria convert it to
toxic products that cause abdominal cramps and diarrhea.
HEALTH BYTE 2: A defect in any of the three enzymes in this pathway causes galactosemia in humans. In
galactokinase deficiency galactosemia, high galactose concentrations are found in blood and urine.
Affected individuals develop cataracts in infancy, caused by deposition of the galactose metabolite galactitol
in the lens. The other symptoms in this disorder are relatively mild, and strict limitation of galactose in the diet
greatly diminishes their severity. Transferase-deficiency galactosemia is more serious; it is characterized by
poor growth in childhood, speech abnormality, mental deficiency, and liver damage that may be fatal, even when
galactose is withheld from the diet. Epimerase-deficiency galactosemia leads to similar symptoms, but is less
severe when dietary galactose is carefully controlled.

19. A 2-week-old newborn was brought to the pediatrician due to
frequent vomiting, lethargy, and diarrhea. Family history revealed
that the child never seemed to eat well, and had only been breast-fed.
Physical examination revealed an enlarged liver and jaundice. The
pediatrician was suspicious of an inborn error of metabolism and
referred the child to an ophthalmologist for a slitlamp exam, the
result of which is shown below. An enzyme that may be defective in
this child is which one of the following?
(A) Fructose-1,6-bisphosphatase
(B) Galactose-1-phosphate uridylyltransferase
(C) Galactokinase
(D) Glycogen synthase
(E) Fructokinase
The answer is B: Galactose-1-phosphate uridylyltransferase.
:The child has classic galactosemia, a defect in galactose-1-phosphate uridylyltransferase. Due to
the accumulation of galactose-1-phosphate, galactokinase is inhibited, and free galactose accumulates
within the blood and tissues. The accumulation of galactose in the lens of the eye provides substrate
for aldose reductase, converting galactose to its alcohol form (galactitol). The accumulation of galactitol
leads to an osmotic imbalance across the lens, leading to cataract formation. Additionally, the increased
galactose-1-phosphate, at very high levels in the liver, blocks phosphoglucomutase activity, resulting in
ineffective glucose production from glycogen (phosphorylase degradation of glycogen will produce
glucose-1-phosphate, but this cannot be converted to glucose-6-phosphate if phosphoglucomutase activity is
inhibited). A defect in galactokinase will lead to nonclassical galactosemia, with cataract formation, but
none of the feeding problems associated with classical galactosemia (associated with the accumulation of
galactose-1-phosphate) are observed in nonclassical galactosemia. None of the other enzymes listed, if
deficient, will give rise to the symptoms produced, particularly cataract formation. A defect in glycogen
synthase would lead to reduced glycogen levels and fasting hypoglycemia. A defect in fructokinase leads to
fructosuria (fructose in the urine), but no overt symptoms of disease. The figure above indicates the pathway
for galactose metabolism and the defects in classical and nonclassical galactosemia.
CASE STUDY:

20. D-Fructose is present in free form in many fruits, and honey. The major dietary
source is sucrose (cane sugar), a disaccharide consisting of glucose and fructose. In
the body, entry of fructose into the cells is not dependent on insulin. This is in
contrast to glucose, which requires insulin for this purpose.
2. Fructose Metabolism
Fructose is phosphorylated by the enzyme fructokinase in the liver, kidney, and
intestine. Fructokinase catalyzes phosphorylation of fructose at C-1 to form
fructose 1-phosphate. The latter is cleaved into dihydroxyacetone phosphate (DHAP)
and glyceraldehyde by the enzyme aldolase B. Defective action of this enzyme leads
to a disorder called hereditary fructose intolerance .
Aldolase B is an isoenzyme of the aldolase (see Reaction 4-glycolysis), and it can
cleave both fructose 1,6-bisphosphate and fructose 1-phosphate. Glyceraldehyde
is phosphorylated by the enzyme triokinase to glyceraldehyde 3-phosphate, which
along with DHAP, is metabolized further by glycolysis or gluconeogenesis.

21. HEALTH BYTE 1: Hereditary fructose intolerance: These are disorders caused by the deficiency of one of the fructose metabolizing enzymes.
The enzymes involved are:
1. Aldolase B: Deficiency of aldolase B results in intracellular accumulation of fructose 1-phosphate. There is vomiting, jaundice,
and hypoglycaemia caused by intracellular accumulation of fructose 1-phosphate. This compound allosterically inhibits liver
phosphorylase to block glycogenolysis, thus leading to hypoglycaemia. Besides tying up phosphate, and thereby impairing
ATP synthesis, fructose 1-phosphate inhibits aldolase and Phosphohexose isomerase; these changes result in liver damage,
and jaundice is commonly seen.
2. Fructose 1,6-bisphosphatase: Deficiency of this gluconeogenic enzyme causes fructose intolerance similar to aldolase-B
deficiency, but these patients also have fasting hypoglycaemia. They can form glucose from stored glycogen, but
gluconeogenesis is blocked. Glycogen degradation can maintain a reasonably normal blood glucose level for many hours, but
the defect in gluconeogenesis results in dangerous hypoglycaemia when the period of fasting exceeds 14–18 hours.
HEALTH BYTE 2: a. Liver damage: The activity of fructokinase far exceeds that of aldolase B, so fructose 1-phosphate tends to accumulate
(concentration in liver can reach up to 10umol/g). This compound allosterically affects several enzymes of carbohydrate
metabolism and also ties up substantial portion of the phosphate in the cell. This can impair oxidative phosphorylation, and lower
the synthesis of ATP from ADP, with consequent damage to liver cells.
b. Hyperlactataemia: This can be traced to a rapid metabolism of fructose to pyruvate, which is in equilibrium with lactate.
c. Hypertriglyceridaemia: Fructose is rapidly metabolized to yield acetyl CoA, which is channeled into lipogenesis (fatty acids
and triglycerides synthesis) in the liver.
d. Hyperuricaemia: This is due to excessive accumulation of ADP and AMP (due to lack of Pi) followed by their degradation to
uric acid.

22. A 3-month-old girl is brought to the pediatrician due
to fussiness and lethargy. According to the parents, the
baby was just fine until the mother needed to return
to work, and the baby was being switched from breast
milk to baby foods, formula, and fruit juices. At that
time, the child cried while feeding, sometimes vomited,
and had been lethargic. The baby’s appetite seemed to
have worsened. The parents thought that if only formula was
used, the baby was better, but they really could not remember.
Which possible enzyme defect might lead to this case
presentation?
(A) Galactokinase
(B) Fructokinase
(C) Aldolase
(D) Hexokinase
(E) Glucokinase
CASE STUDY:
The answer is C: Aldolase. The disorder is hereditary fructose intolerance, with a reduced ability to convert fructose-1-phosphate to
dihydroxyacetone phosphate and glyceraldehyde. The specific defect is in aldolase B, with its activity reduced by as much as 85%.
This problem is only evident when sucrose is introduced into the diet, and fructose enters the liver. The accumulation of fructose-1-
phosphate, due to the reduced aldolase activity, leads to a constellation of physiological problems resulting in nausea, vomiting, and
hypoglycemia. Elimination of fructose from the diet will reverse the symptoms.
Galactokinase is needed for galactose metabolism; since the patient digests milk normally, galactokinase activity is not altered. Similarly,
glucose metabolism is not adversely affected (milk contains lactose, which is split into glucose and galactose), indicating that hexokinase
and glucokinase activities are normal. The defect in aldolase B will hinder glycolysis, but the liver also contains aldolase C activity (this
isozyme will not split fructose-1-phosphate), which enables glucose metabolism to be very close to normal.
A deficiency in fructokinase will lead to an accumulation of fructose (not fructose-1-phosphate), which is released into the urine
(fructosuria), but does not lead to the physiological symptoms exhibited by the patient. The fructose pathway (indicating the reaction
catalyzed by aldolase B), and its relationship to glycolysis, is shown below.

23. Clinical significance of Uronic acid pathway.
A. Glucuronic acid is used for synthesis of mucopolysacharides,
detoxification, conjugation with bilirubin, steroid hormone etc.
In plants and mammals other than man Vit. C is synthesized from gulonate by
this pathway. This pathway utilizes glucuronic acid of endogenous origin for
energy production. Dietary xylitol is utilized by this pathway.
Uronic/Glucuronic acid Pathway

24. Write about essential pentosuria
A. This inherited disease is characterized by excretion L-xylulose in urine. It is due
to deficiency of enzyme xylitol dehydrogenase. Due to lack of this enzyme L-
xylulose cannot be converted to xylitol and it accumulates in blood and get
excreted in urine.
Drugs like barbiturate and paracetamol increases utilization of glucose by this
pathway.

25. Glycogen Storage Diseases

27.  Textbook of Biochemistry-DM Vasudevan
 Textbook of Biochemistry-U Satyanarayana
 Nelson, D. L., & Cox, M. M. (2017). Lehninger principles of biochemistry (7th ed.).
W.H. Freeman.
Thank You