Biochemistry, Molecular Biology, Physiology, Microbiology, Immunology, Pharmacology & Drug Discovery
Preparation Lecture Notes, Multiple Choice Questions, Scientific Discoveries

Homocystinuria & Homocysteinemia: Clinical Condition and Biochemical Interpretation

August 29, 2018
Homocystinuria is an inherited disorder primarily caused by a deficiency of enzymes of methionine metabolism which lead to accumulation and excretion of homocystine in the urine. Cysteine is a non-essential amino acid that is synthesized from methionine and involves four steps catalyzed by four different enzymes as shown in figure 1. Defective enzymes of this pathway or deficiency of co-factors or enzymes that are involved in the recycling of the co-factors lead to elevated blood homocysteine and homocystinuria. The increased blood homocysteine is also known as homocysteinemia are associated with various cardiovascular diseases and stroke but the mechanism is not unknown.

Metabolism of Methionine and Cysteine 

Homocystinuria can be classified into three different categories based on the defective enzymes and symptoms:
Homocystinuria type I
Homocystinuria type II
Homocystinuria type III

Homocystinuria type I:
The classical homocystinuria is an autosomal recessive disorder that involves multiple organ involvement including the eye, skeletal system, thromboembolism, delayed development, and intellectual disability. The CBS enzyme is located on the long arm of chromosome 21 (21q22.3) and the mutation of the CBS gene may result in reduced activity of cystathionine beta-synthase resulting in classical homocystinuria. The confirmatory diagnosis includes increased plasma methionine, homocysteinemia, and presence of homocystine in the urine.

Homocystinuria type II & II
Methylcobalamin is required for the reconversion of homocysteine to methionine and the defect in the formation of methylcobalamin or methylation pathway results in type II & III homocystinuria. The type II & III homocystinuria is characterized by megaloblastic anemia, homocystinuria and low levels of methionine with mild clinical severity.

Possible Causes of Homocystinuria and Homocysteinemia
Biochemical Diagnosis:
-Nitroprusside test
-Amino Acid Chromatography in Urine
-Enzyme activity in fibroblast and Mutation analysis

The treatment of homocystinuria may include dietary control of proteins and amino acids, supplementation of cyanocobalamin, folic acid and pyridoxine may alleviate the symptoms.
Homocystinuria & Homocysteinemia: Clinical Condition and Biochemical Interpretation  Homocystinuria & Homocysteinemia: Clinical Condition and Biochemical Interpretation Reviewed by Biotechnology on August 29, 2018 Rating: 5

Glycogen synthesis and Breakdown Pathway

August 22, 2018
Glycogen is a polysaccharide consist of glucose linked together by glycosidic linkage. In animals and humans; glucose is stored in the form of glycogen in the liver (~10%) and muscles (~2%). These stored glycogen molecules can readily be degraded into glucose molecules and enter into the glycolytic pathway for energy. Liver glycogen can also contribute to the maintenance of normal blood glucose.  The glycogen synthesis and breakdown pathway are highly regulated and, the synthesis and breakdown do not occur at the same time. 


Figure 1: Overview of Glycogen synthesis (Glycogenesis)

Synthesis of Glycogen
The addition of glucose to form glycogen requires a primer molecule where the glucose can be added to the non-reducing ends. During de novo synthesis, glucose molecules are added to tyrosine residues of primer protein glycogenin. The enzyme Glycogen synthase catalyzes the addition of glucose molecules at the nonreducing end of core glycogen molecule  In this reaction, an activated UDP-glucose molecule forms 1-4 glycosidic linkage with existing glucose moiety of glycogen molecule and free UDP is liberated. This results in the elongation of a glycogen molecule with the addition of one glucose moiety in each reaction. 

Figure 2: Glycogen Synthase-addition of glucose from UDP-glucose to core glycogen molecules.


Figure 3: Overview of Glycogen Breakdown(Glycogenolysis)

Figure 4: Glycogen phosphorylase-removal of glucose from glycogen molecules.

Regulation of Glycogen Metabolism

-Glycogen synthesis occurs when the glucose and ATP are abundant in the cells. In contrary, glycogen breakdown release glucose for muscle contraction and regulation of blood glucose. 

-Glycogen metabolism is regulated by allosteric modification and covalent modifications. 

-The glycogen synthesis and breakdown are reciprocally regulated to ensure that both pathways do not occur at the same time in the cell.

Glycogen phosphorylase regulation

Allosteric modification: 

-The glycogen phosphorylase exists in two different conformations. T-state or inactive state and R-state or active state. 
-In muscle, the binding of an AMP molecule to glycogen phosphorylase enzyme shifts the T-state glycogen phosphorylase to R-state. 
-ATP and Glucose-6-phosphate exert an inhibitory effect by favoring the T-state of glycogen phosphorylase (inactive form).
-In the liver, the presence of glucose shifts the R-state glycogen phosphorylase to T-state (inactive). 

Allosteric Modification of glycogen phosphorylase in muscle 

Allosteric Modification of glycogen phosphorylase in liver

Covalent modification:
-Insulin and glucagon reciprocally regulate glycogen phosphorylase by adding/removing phosphate group to the enzyme glycogen phosphorylase. 
- During muscle contraction, in addition to low glucose and ATP, the release of calcium from sarcoplasmic reticulum activates calcium-dependent protein kinase that inturns phosphorylate and activate glycogen phosphorylase. 
- Calcium ions partially activate calcium-dependent protein kinase that requires phosphorylation by protein kinase A for full activity. Epinephrine binding to its receptor induces the signaling cascade to activate protein kinase A. 
- In the liver, glucagon and epinephrine induce the signaling cascade to phosphorylate activate phosphorylase kinase and activate downstream protein glycogen phosphorylase.
-In response to high blood glucose concentration, insulin is released from beta cells of the pancreas.
-Insulin binds to tyrosine kinase receptors and induces the signaling pathway to activate protein phosphatase 1. The protein phosphatase 1 catalyzes the removal of the phosphate group from glycogen phosphorylase and deactivates it. 


Phosphorylation activates glycogen phosphorylase enzyme and increases glycogen breakdown in exercising muscle and liver when blood glucose is low. 


Dephosphorylation inactivates glycogen phosphorylase enzyme and decreases glycogen breakdown in resting muscle and liver when blood glucose is abundant. 

Glycogen synthase regulation
Covalent Modification:
-The glycogen synthase is the regulatory enzyme of glycogen synthesis.
-Insulin induces glycogen synthesis by activating the enzyme glycogen synthase (in a dephosphorylated state).
- In contrast, glucagon and epinephrine deactivate the enzyme (increasing phosphorylation) thereby reducing glycogen synthesis. 


Dephosphorylation activates glycogen synthase enzyme and increases glycogen synthesis in resting muscle and liver when blood glucose is abundant. 


Phosphorylation inactivates glycogen synthase enzyme and decreases glycogen synthesis in exercising muscle and liver when blood glucose is low. 

Glycogenesis is the biosynthetic pathway for synthesis of glycogen from glucose molecules.  This biosynthetic pathway can be divided into two stage i.e activation of glucose and addition of glucose to core glycogen molecules at a nonreducing end.

Activation of glucose molecules: 
The precursor glucose molecules are first activated by an enzyme hexokinase/glucokinase to form glucose-6-phosphate. The next step is the conversion of glucose-6-phosphate to glucose-1-phosphate which is catalyzed by enzyme phosphoglucomutase. This enzyme catalyzes the transfer of phosphate group 6-carbon group to 1- carbon resulting in glucose-1-phosphate. 

The second reaction is the formation high energy UDP-glucose catalyzed by an enzyme UDP-Glucose pyrophosphorylase. In this reaction, the uridine monophosphate group from UTP to form UDP-glucose and pyrophosphate. The pyrophosphate formed is subsequently hydrolyzed to inorganic phosphates to release energy. 

The glycogen molecule is highly branched. The branching glycogen molecule is introduced by branching enzyme that transfers the oligopeptide glucose moieties from 1-4 glycosidic linkage to form 1-6 glycosidic linkage with the interior glucose moiety of glycogen molecule. This results in branching of glycogen molecule at every 8-10 residues and compact helical structure.

Glycogenolysis is the breakdown of glycogen to glucose-6-phosphate and involve a series of enzyme catalyzed reactions.

Phosphorolysis of Glycogen to glucose-1-phosphate
The first reaction of glycogen breakdown is the phosphorolysis of glycogen molecule to liberate one glucose-1-phosphate. This phosphorolysis reaction is catalyzed by a pyridoxal-5-phosphate requiring enzyme Glycogen phosphorylase. During this reaction, the inorganic phosphate is incorporated to the glucose molecules allowing the intermediates to directly enter other metabolic pathways (glycolysis and pentose phosphate pathway)

Conversion to Glucose-6-phosphate
Glucose-1-phosphate is converted into glucose-6-phosphate catalyzed by an enzyme phosphoglucomutatse as described above. In liver, glucose-6-phosphate formed are converted into free glucose by enzyme glucose-6-phosphatase and contributes to blood glucose maintenance. In contrast, muscle tissue lacks an enzyme glucose-6-phosphatase and glucose-6-phosphate produced in the muscle tissues do not contribute to the blood glucose maintenance. The function of glycogen in muscle is to feed glucose-6-phosphate into glycolytic pathway for ATP required for muscle contraction during exercise. 

(Note: The hydrolysis to glucose during this step would have required ATP for the activation. Therefore, the phosphorolysis captures the inorganic phosphate and conserve one ATP in energy-deprived cells.  that may would have required if the hydrolysis of glycogen to glucose.)

Removal of glucose by debrancing enzyme
As the glycogen are highly branched, and glycogen phosphorylase only removes glucose from nonreducing end of 1-4 glycosidic linkage; an additional enzyme 1,6-glucosidase  (debranching enzyme) hydrolyzes the 1-6 glycosidic linkage at branch site release free glucose. 

Glycogen synthesis and Breakdown Pathway Glycogen synthesis and Breakdown Pathway Reviewed by Biotechnology on August 22, 2018 Rating: 5

Amino Acid Metabolism and Aminoacidurias : MCQ

August 21, 2018
Glutamate and Glutamine Metabolism
Glutamate and Glutamine are nutritionally non-essential amino acids that are the building block of proteins and, the precursor for the synthesis of other nonessential amino acids, urea, and neurotransmitters. Glutamine also serves as co-substrate for the formation of asparagine. The glutamate and glutamine synthesis reaction uniquely incorporate free ammonium ions and is important for ammonia metabolism and detoxification.

The metabolic pathway for the synthesis of glutamate and glutamine involves i) Conversion of alpha-ketoglutarate to glutamate by enzyme glutamate dehydrogenase, an NADPH requiring an enzyme that incorporates free ammonia into alpha-keto-glutarate. ii) Conversion of glutamate to glutamine is catalyzed by Glutamine synthetase which incorporates ammonium ion and utilizes ATP as energy.

Metabolism of Glutamine and Glutamine 

Fates of Glutamate 

Metabolism of Cysteine
Metabolism of Methionine and Cysteine 

Metabolism of Serine and Glycine

Overview of Glycine and Serine metabolism

Multiple Choice Questions

1) Glutamate and Glutamine are nutritionally non-essential amino acids. The conversion of alpha-ketoglutarate to glutamate and glutamine are catalyzed by:
a) Glutamate synthetase and Glutamate dehydrogenase
b) Glutamate transaminases
c) Glutamate dehydrogenase and Glutamine synthetase
d) None of Above

2) Which of the following statement is NOT TRUE:
a) Glutamate dehydrogenase catalyzes the incorporation free ammonium ion to form glutamate
b) Glutamate synthetase utilized ATP as energy for the incorporation of ammonium ion to form glutamine
c) Asparagine synthetase utilized ATP as energy for the incorporation of ammonium ion to form asparagine
d) Asparagine dehydrogenase catalyzes the incorporation free ammonium ion to form aspartate

3) Glutamate and Glutamine are a critical entry point for free ammonia to enter the metabolic pathway. Which of the following pathway glutamate metabolism is regulated by allosteric inhibitors such as alanine, glycine, carbamoyl phosphate
a) Glutamate synthase
b) Glutamate synthetase
c) Glutamate dehydrogenase
d) Glutamate deoxygenase

4) N-acetyl Glutamate is an activator of urea synthesis and formed from condensation glutamate and acetyl CoA. Which of the following enzyme catalyzes this reaction?
a) Arginosuccinate synthetase
b) Carbamoyl phosphate synthetase
c) N-Acetyl glutamate synthase
d) Glutaminase

5) Hydroxyproline and hydroxylysine are formed by hydroxylation of proline and lysine; the reaction catalyzed by prolyl and lysyl hydroxylase. Which of the following serve as coenzyme?
a) Biotin
b) Thiamine
c) Ascorbate

d) Niacin

6) Cysteine is a nutritionally non-essential amino acid, and it is synthesized from
a) Proline
b) Valine
c) Glycine
d) Methionine

7) 3-phosphoglycerate is the precursor for all of the following amino acids EXCEPT
a) Serine
b) Glycine
c) Alanine
d) Cysteine

8) The conversion of serine to glycine is catalyzed by an enzyme serine hydroxymethyltransferase. The co-substrate required for this reaction is:
a) Biotin
b) Pyridoxal Phosphate
c) Tetrahydrobiopterin
d) Tetrahydrofolate

9) Alpha-ketoglutarate is the precursor for all the following amino acid except
a) Proline
b) Arginine
c) Glutamine
d) Asparagine

10) Which one of the following statement concerning a one-week-old male infant with undetected classic phenylketonuria is correct?
a) Tyrosine is a non-essential an amino acid for the infant
b) High levels of phenylpyruvate appear in the urine
c) A diet devoid of phenylalanine should be initiated immediately
d) Therapy must begin within the first year of life.

11) Maple syrup disease is a disorder due to deficiency of pathway that degrades:
a) Tyrosine
b) Tryptophan
c) Leucine
d) Methionine

12) The phenylalanine metabolism is blocked in the metabolic disease phenylketonuria. Which of the following product is formed during normal metabolism of phenylalanine by phenylalanine hydroxylase?
a) Tyrosine
b) Phenylpyruvate
c) Phenylacetate
d) Phenyl lactate

13) Identify both glucogenic and ketogenic amino acid.
a) Methionine
b) Cysteine
c) Tryptophan
d) Valine

14) Identify the amino acid that participates in methyl group transfer:
a) Leucine
b) Valine
c) Lysine
d) Methionine

15) Which of the following amino acids have an important role in the transport of amino group from peripheral tissues to the liver?
a) Serine
b) Methionine
c) Glutamine
d) Arginine

16) The following metabolite is excreted via urine to maintain the nitrogen balance except:
a) Amino acids
b) Creatinine
c) Urea
d) Ammonia

Multiple Choice Answers
1-c) Glutamate dehydrogenase and Glutamine synthetase
2-d) Asparagine dehydrogenase catalyzes the incorporation free ammonium ion to form aspartate
3-b) Glutamate synthetase
4-c) N-Acetyl glutamate synthase
5-c) Ascorbate
6-d) Methionine
7-b) Glycine
8-c) Tetrahydrobiopterin
9-d) Asparagine
10-b) High levels of phenylpyruvate appear in the urine
11-c) Leucine
12-a) Tyrosine
13-c) Tryptophan
14-d) Methionine
15-c) Glutamine
16-a) Amino acids

Amino Acid Metabolism and Aminoacidurias : MCQ Amino Acid Metabolism and Aminoacidurias : MCQ Reviewed by Biotechnology on August 21, 2018 Rating: 5

Reciprocal Regulation of Glycolysis and Gluconeogensis Prevent Futile Cycle

August 20, 2018
Similar to most of the metabolic pathways, glucose synthesis and breakdown is regulated by three different mechanisms:
a) Allosteric regulators
b) Covalent modification
c) Changes in gene expression

Regulation of glycolytic pathway:
As described in the previous page and figure 1, glycolysis is regulated by three irreversible enzymes namely: Hexokinase/glucokinase, Phosphofructokinase, and Pyruvate kinase.

Figure 1: Allosteric Regulators of Glycolysis and Gluconeogenesis 

Hexokinase is a ubiquitously expressed enzyme that set the pace of glycolysis. Hexokinase has a high affinity for glucose and transfers negatively charged phosphate group to a glucose molecule. This step traps the glucose inside the cells and funnels into various metabolic pathways. The high concentration of glucose-6-phosphate signals that the cell no longer requires for energy or other biosynthetic pathways, and inhibit enzyme hexokinase.

Glucokinase is expressed in tissues(e.g. liver, pancreas) that play an important role in maintaining glucose concentration in blood. Glucokinase differs from hexokinase in two aspects: i) Glucokinase has low affinity for glucose with Km above normal blood glucose concentration ii) Glucose-6-phosphate has no inhibitory effect on glucokinase. The low affinity and high catalytic activity of glucokinase are suited for its function in the liver. When the blood glucose is high, glucokinase rapidly binds and converts glucose to glucose-6-phosphate. In contrast, when blood glucose is limited, glucokinase has low activity and allows glucose to be distributed into tissues such as the brain and red blood cells.

Figure 2: Glucokinase and Hexokinase differ the affinity of the substrate. This is an example of how isoform regulates the metabolic pathway in different tissue

The phosphofructokinase is the second irreversible step of glycolysis which is regulated by various allosteric effector molecules. When ATP concentration is high in cells, ATP binds to the allosteric site and inhibit the enzyme activity of PFK. In contrast, AMP reverses the inhibitory action of ATP. Citrate and hydrogen ion also inhibit phosphofructokinase. Citrate is metabolic intermediates formed in the TCA cycle. The abundance of citrate signals the abundance the metabolic intermediates and energy equivalents in the cell. The high hydrogen concentration in muscle tissues inhibits the glycolysis by inhibiting the phosphofructokinase.

In the liver, fructose-2,6-bisphosphate acts as an allosteric activator of PFK. Fructose-1,6-bisphosphate binds and activates PFK by decreasing the inhibitory effect of ATP. When fructose-6-phosphate is high, phosphofructokinase-2 (an isoform of PFK) phosphorylate it to form fructose -2,6-bisphosphate. PFK-2 is a bifunctional enzyme that has both the kinase domain and the phosphatase domain. When fructose-6-phosphate is low, phosphatase domain of PFK-2 catalyzes the hydrolysis of fructose-2,6-bisphosphate to form fructose-6-phosphate. The kinase and phosphatase domain of PFK-2 is regulated by covalent modification. In response to glucagon, increased cAMP signals the activation of protein kinase A, phosphorylation of PFK2 and activation of phosphatase domain. In contrast, insulin activates protein phosphatase that in turn dephosphorylate PFK-2 thereby activating PFK domain.

Pyruvate Kinase
The different isoforms of pyruvate kinase are expressed in different tissues and they are regulated differently. The L-type isoform of pyruvate kinase that is expressed in the liver is regulated by covalent modification. The phosphorylated state is inactive and dephosphorylated enzyme is active. High blood glucose leads to activation of pyruvate kinase whereas low blood glucose inactivates pyruvate kinase. In contrast, M-type pyruvate kinase isoform expressed in muscle and brain is not sensitive to regulation by covalent modification. T Both L and M type isoforms are regulated by allosteric modifiers such as ATP, citrate, and Fructose-1,6-bisphosphate as shown in figure 1.
Reciprocal Regulation of Glycolysis and Gluconeogensis Prevent Futile Cycle Reciprocal Regulation of Glycolysis and Gluconeogensis Prevent Futile Cycle Reviewed by Biotechnology on August 20, 2018 Rating: 5

Pentose Phosphate Pathway: Source of NADPH for Reductive Biosynthesis

August 20, 2018
Introduction to Pentose Phosphate Pathway
The pentose phosphate pathway (Hexose monophosphate pathway) is the metabolic pathway that occurs in the cytosol and generates NADPH that is utilized in various biosynthetic pathways. This pathway can be broadly classified into two categories i.e oxidative steps and non-oxidative steps. In the oxidative stage of pentose phosphate pathway, the glucose-6-phosphate is converted to five carbon ribulose-5 phosphate and generation of NADPH. During the non-oxidative stage, the interconversion of pentose sugars and hexose sugars are catalyzed by isomerases and aldolases. The fate of these reversible steps depends on the anabolic and energy status of the cells. The two main functions of the pentose phosphate pathway are
a) Provide NADPH for biosynthetic pathways
b) Provide pentose sugars for nucleotide synthesis

Oxidative Stage of Pentose Phosphate Pathway
The oxidative stage of pentose phosphate pathway is a stepwise oxidation-reduction reaction followed by decarboxylation.
-The first step of the pentose phosphate pathway is the conversion of glucose-6-phosphate to 6-phosphogluconolactone and generation of NADPH from NADP+. This oxidation-reduction coupled reaction is catalyzed by enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD is a rate-limiting enzyme of the pentose phosphate pathway.
-The second step is the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate catalyzed by enzyme gluconolactone hydrolase.
-The third enzyme 6-phosphogluconate dehydrogenase catalyzed an oxidation-reduction reaction of pentose phosphate pathway to yield 3-keto-6-phosphogluconate which is non-enzymatically decarboxylated to form ribulose-5-phosphate. During this conversion, the second NADPH is generated. Altogether, the conversion of glucose-6-phosphate to ribulose-6-phosphate yields two NADPH which is used for various biosynthetic and physiological processes of the cells.

Figure 1: Oxidative Stage of the Pentose Phosphate Pathway.

1) Glucose-6-phosphate dehydrogenase; 2) Gluconolactone hydrolase; 3) 6-phosphogluconate dehydrogenase; 4) nonenzymatic decarboxylation

Non-oxidative Stage of Pentose Phosphate pathway:
Once the ribulose-5-phosphate is formed, the series of reversible steps can result in the formation of six sugars. Transketolase and transaldolases are the enzymes that catalyze a series of reversible reaction to convert pentose sugar into fructose-6-phosphate which can again re-enter glycolysis or pentose phosphate pathway depending on the cellular demand of ATP and NADPH.
-First, the ribulose-5-phosphate is converted into xylulose 5-phosphate and ribose-5-phosphate catalyzed by enzyme 3-epimerase and keto-isomerase respectively.
-Second, the xylulose-5-phosphate and ribose-5-phosphate are converted into glyceraldehyde-3-phosphate and sedoheptulose by enzyme Transketolase. Transketolase is a thiamine contained enzyme that transfers 2 carbons from keto-sugars to aldo-sugar (or vice-versa).

Figure 2: Non-oxidative Stage of the Pentose Phosphate Pathway

-Third, the glyceraldehyde-3-phosphate and sedoheptulose are converted to fructose-6-phosphate and erythrose-4-phosphate catalyzed by enzyme Transaldolase. Transaldolase transfers 3 carbons from keto-sugar to aldo-sugar.
-Lastly, another 2 carbons from xylulose-5-phosphate (keto-sugar) to erythrose-4-phosphate to yield fructose-6-phosphate and glyceraldehyde.
Overall, three pentose sugars are utilized to form 2 fructose-6-phosphate and 1 glyceraldehyde-3-phosphate.

Cellular demand for ATP, NADPH, and pentose sugar modulate pentose phosphate pathway
-Scenario 1: When ribose-5-phosphate and NADPH are the prime need of the cells (especially dividing cells), most of the glucose-6-phosphate is converted into pentose sugar and NADPH. The oxidative reaction is most active during this state of cells.

-Scenario 2: When NADPH is required for other biosynthetic pathways, the pentose sugars are converted into fructose-6-phosphate which can be re-converted into glucose-6-P by glycolytic enzyme phosphoglucoisomerase. The glucose-6-phosphate thus formed re-enters into the pentose phosphate pathway. In this mode, one glucose-6-phosphate can generate 12 NADPH (please derive how !!!).

-Scenario 3: When NADPH and ATP are required (for example erythrocytes) 3 glucose-6-phosphate is converted into pentose phosphate pathway and generate 6 NADPH. The 3 pentose sugar formed during the oxidative stage is converted into 2 fructose-6-phosphate and 1 glyceraldehyde-3-phosphate. These can enter glycolysis to form additional ATPs and NADH.
Pentose Phosphate Pathway: Source of NADPH for Reductive Biosynthesis Pentose Phosphate Pathway: Source of NADPH for Reductive Biosynthesis Reviewed by Biotechnology on August 20, 2018 Rating: 5

What are the precursors of gluconeogenesis?

August 20, 2018
The gluconeogenesis is the formation of glucose from a non-glucose precursor including pyruvate, lactate, and amino acids.

Pyruvate and Lactate:
Pyruvate is the end product of glycolysis which can be further metabolized to either acetyl CoA or lactate. The conversion of pyruvate to these products depend on tissue-type, ATP status, and regulatory effector molecules. The conversion of pyruvate to acetyl CoA is an irreversible step that is catalyzed by enzyme pyruvate dehydrogenase that occurs mainly in oxidative tissues such as liver, cardiac muscle. In contrast, the conversion of pyruvate to lactate is reversible step catalyzed by lactate dehydrogenase that occurs mainly in non-oxidative tissues such as red blood cells. The lactate dehydrogenase is also present in skeletal muscle that converts pyruvate to lactate during vigorous exercise.

Figure 1: Fates of Pyruvate PDH- Pyruvate dehydrogenase, LDH- Lactate dehydrogenase

Pyruvate and Lactate formed in peripheral tissues shuttled into the liver for gluconeogenesis:
The lactate formed during anaerobic glycolysis can directly be shuttled into the liver. In the liver, the lactate dehydrogenase isoenzyme converts lactate and pyruvate. Alternately, the lactate formed during anaerobic glycolysis is reconverted into pyruvate and subsequently converted to alanine by enzyme alanine transaminase. The lactate or alanine diffuse into the bloodstream and taken up by the liver. In the liver, alanine is converted by liver alanine transaminase to convert into pyruvate.

Figure 2: Glucose-alanine cycle

Figure 3: Cori cycle in Erythrocytes

Amino acid
Glucogenic amino acids are the group of amino acids whose metabolic intermediates can enter into the gluconeogenic pathway and form glucose. The examples include alanine, aspartate, glutamate etc.

Propionyl CoA
Propionyl CoA an intermediate of odd-chain fatty acids are also the precursor for the gluconeogenic pathway. Propionyl CoA is converted into methyl malonyl CoA by enzyme propionyl CoA carboxylase ( a biotin-containing enzyme). The methyl malonyl CoA is converted into succinyl CoA by enzyme methyl malonyl CoA mutase and the succinyl CoA enters TCA cycle to form oxaloacetate. The oxaloacetate thus formed enters the gluconeogenic pathway to form glucose.
What are the precursors of gluconeogenesis? What are the precursors of gluconeogenesis? Reviewed by Biotechnology on August 20, 2018 Rating: 5

Pharmacological Inhibitor of Purine and Pyrimidine Metabolism

August 19, 2018
Pharmacological agents that directly or indirectly inhibit purine and pyrimidine synthesis have been used as anti-bacterial agents and anti-tumor drugs. These class or drugs inhibit purine/pyrimidine synthesis and availability in the cells thereby decreases the cell replication. The inhibitors of purine degradation pathway are used for the treatment of gout.

Anti-tumor agents
Fluorouracil is a mechanism based drug that inhibits the enzyme Thymidylate synthetase (TS). Thymidylate synthetase is an enzyme that catalyzes the synthesis of deoxyuridine monophosphate to deoxythymidine monophosphate, a building block of DNA. The inhibitor of the enzyme led to decreased production of dTMP and reduced substrate for DNA replication and cell cycle. 5-Fluorouracil competes with uracil to bind enzyme TS. 5-fluorouracil is metabolically converted to 5-FdUMP which covalently binds to the enzyme and inactivates it. These types of inhibitors that competitively binds and irreversibly inhibit the enzymes are known as suicide inhibitors.

Thymidylate synthetase along other enzymes of purine metabolism utilizes folic acid for one carbon transfer reaction. The structural analogs of folate such as methotrexate that inhibits the enzyme Dihydrofolate reductase also serves as an anti-cancer drug. The inhibition of dihydrofolate reductase decreases the recycling of dihydrofolate to tetrahydrofolate, an active 1-carbon acceptor molecule.

Antibacterial agents:
Sulfonamides and Trimethoprim are the selective inhibitors of bacterial enzymes. Sulfonamides are the structural analogs of para-aminobenzoic acid that competitively inhibit bacterial synthesis of folic acid synthesis in bacteria. Trimethoprim inhibits the recycling of dihydrofolate to tetrahydrofolate by selectively inhibiting bacterial dihydrofolate reductase.

Treatment of Gout:
Gout is a disorder associated with the deposition of uric acid crystal in soft tissues and joints of extremes. The accumulation of uric acid crystal in joints may lead to the inflammation, macrophage recruitment and result in for the formation of topi and gouty arthritis. In gout, the uric acid accumulation may be associated with either increased production or decreased excretion through the kidney. Allopurinol is the pharmacological agent that decrease the production of uric acid by inhibiting a key enzyme Xanthine Oxidase of its pathway.

Pharmacological Inhibitor of Purine and Pyrimidine Metabolism Pharmacological Inhibitor of Purine and Pyrimidine Metabolism Reviewed by Biotechnology on August 19, 2018 Rating: 5

Lesch Nyhan Syndrome: Clinical Presentation and Biochemical Diagnosis

August 09, 2018
A 3-year-old child was brought to the hospital with a complaint of self-mutilation. He had a chronic ulcer on the buccal surface of lips, and self-inflicted trauma with biting his finger. History revealed that severe motor retardation was apparent by 6 months and he has never been able to lift or support his trunk. The growth chart showed that he has growth retardation. Biochemical analysis was performed which are represented below:

Serum Uric acid: 9.0 mg/dL
Blood Urea : 32 mg/dL
Serum Sodium : 139 mmol/l
Potassium: 5.1 mmol/l
Calcium 39 mmol/l
Total Protein: 70 g/L
Albumin: 23 g/L
Urinary Uric acid: 160mg/100 ml
Urinary Glucose: Absent
Urinary Protein: Absent
Microscopic urine examination: triphosphate crystals

Provisional Diagnosis: Based on the behavioral pattern of self-mutilation, growth retardation, dystonia and increased serum and urinary uric acids are suggestive of Lesch-Nyhan Syndrome.

Biochemical Basis of Lesch-Nyhan Syndrome
Lesch-Nyhan Syndrome (LNS) is an X-linked inborn error of metabolism caused by mutation of the gene HPRT1 encoding enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). HGRT enzyme catalyzes the recycling of purine bases and lack of this enzyme lead to decreased recycling and increase degradation of purines to uric acid. In addition, lower cellular GMP level increases the denovo synthesis of purines (Figure 1). Therefore, increase synthesis and degradation of purines are responsible for the aggravated level of uric acids in these patients and also result in nephrolithiasis, gouty arthritis, and subcutaneous tophi.

Figure 1: Overview of Purine Metabolism and Biochemical Basis of Lesch-Nyhan Syndrome.a) In LNS, the reduced activity of HPRT results in the decreased salvage pathway of purine nucleotide biosynthesis and cellular level of AMP/GMP. b) The reduced cellular AMP/GMP activates the denovo synthesis of purines. c) The overproduction of purines results in increased formation of uric acid that is deposited as crystals in various soft tissues.

These patients also exhibit a distinctive neurobehavioral phenotype, characterized by dystonia, attentional deficits, and behavioral disturbances including self-injury, presumably attributable to dysfunction of the basal ganglia dopamine system. However, it is unclear how a shortage of hypoxanthine phosphoribosyltransferase 1 causes neurological and behavioral problems characteristic of Lesch-Nyhan syndrome.

Genetics and Diagnosis of Lesch-Nyhan Syndrome
Human HPRT is encoded by a single structural gene spanning approximately 45 kb on the long arm of the X chromosome at Xq26 and consists of nine exons with a coding sequence of 654 bp. In LNS, the high heterogeneity of mutations is observed within the HPRT1 gene including deletions, insertions, duplications, and point mutations. Till date more than 300 disease-causing mutations in HPRT1 gene have been reported.

For the diagnosis of Lesch-Nyhan syndrome, the serum, and urinary uric acid are evaluated. With elevated serum and urinary uric acid level, the patients are subjected to genetic testing for characterization of HPRT1 gene mutation and HPRT enzyme activity. The pathogenic mutation in the HPRT1 gene and lower HPRT enzyme activity confirms the diagnosis of Lesch-Nyhan Syndrome. HPRT1 gene is a constitutively expressed in peripheral blood cells and, biochemically diagnosed by a null HPRT activity in erythrocytes. The presence of HPRT mRNA expression and molecular diagnosis are performed using HPRT complementary DNA (including 3′ and 5′ regions) sequencing and genomic DNA sequencing analysis. In some cases, the normal coding region with a reduced expression requires quantification of mRNA using real-time PCR. Prenatal diagnosis for Lesch–Nyhan syndrome may be performed in amniotic cells obtained by amniocentesis at about 15–18 week's gestation, or chorionic villus cells obtained at about 10–12 week's gestation. Both HPRT enzymatic assay and molecular analysis for the known disease-causing mutation are performed.

Lesch Nyhan Syndrome: Clinical Presentation and Biochemical Diagnosis Lesch Nyhan Syndrome: Clinical Presentation and Biochemical Diagnosis Reviewed by Biotechnology on August 09, 2018 Rating: 5
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