Category Archives: Biochemistry

Biochemistry

Enzymes: Structure, Functions, and Classification

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Enzymes are biological catalysts with extraordinary catalytic power. They are central to every biochemical process.  

We know living systems derive energy from the surroundings through biochemical processes. For example, the oxidation of sucrose to CO2 and H2O generates a high amount of power, which we utilize to move, taste, see, and think.  

Chemical reactions like sucrose oxidation cannot happen in the correct time frame and thus cannot sustain life. Hence, as biological catalysts, enzymes play a significant role in biochemical processes. 

Enzymes are highly specific and function in aqueous solutions under very mild conditions of temperature and pH. They act in organized sequences and catalyze hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform chemical energy, and form biological macromolecules from simple precursors.  

Measurements of enzymes in erythrocytes, plasma, and tissues help diagnose some diseases and deficiencies, especially inheritable genetic disorders. They are also critical practical tools in medicine, the chemical industry, food processing, and agriculture.  

General Properties of Enzymes

  1. All enzymes are proteins except for a small group of catalytic RNA molecules.
  2. The integrity of the enzyme’s native protein conformation affects its catalytic activity. Catalytic activity is usually lost if an enzyme is denatured or dissociated into its subunits. 
  3. If an enzyme is broken down into component amino acids, its catalytic activity is permanently destroyed. Thus, protein enzymes’ primary, secondary, tertiary, and quaternary structures are essential to their catalytic activity.
  4. Enzymes affect reaction rates but not equilibria.
  5. These are not consumed in overall reactions. 
  6. Enzymes are required in a few amounts during chemical reactions. 
  7. These have active sites where the interaction with the substrate occurs.
  8. Enzymes are extremely specific to their substrate. 

Structure of Enzymes

As discussed earlier, most enzymes are proteins. Like other proteins, enzymes have molecular weights ranging from approximately 12,000 to greater than 1 million. 

Enzymes have a specific three-dimensional structure that enables them to bind to substrates and conduct chemical reactions.General structure of enzymes may be similar to the primary, secondary, tertiary, or quaternary structure of proteins. Active site is present in all the enzymes and regulatory modification can occur in some enzymes.

Primary Structure

This refers to the linear sequence of amino acids in the enzyme’s polypeptide chain. The sequence is determined by the gene encoding the enzyme and is crucial for its overall structure and function.

Secondary Structure

Enzymes often exhibit secondary structures such as alpha-helices and beta-sheets. These structures result from hydrogen bonding between amino acids in the polypeptide chain. The secondary structure helps determine the overall folding of the enzyme.

Tertiary Structure

It is the three-dimensional arrangement of the entire polypeptide chain, including the secondary structural elements. This structure is stabilized by various interactions such as hydrogen bonds, disulfide bonds, hydrophobic interactions, and electrostatic interactions between amino acid side chains.

Quaternary Structure (if applicable)

Some enzymes consist of multiple polypeptide chains, and their quaternary structure refers to the arrangement of these subunits. The interactions between subunits can also contribute to the enzyme’s stability and function.

Active Site

This is a crucial feature of enzyme structure where the substrate binds, and the catalytic reaction occurs. The active site is typically a small crevice or pocket on the enzyme’s surface that is similar in shape and chemical properties to the substrate molecules. It often involves specific amino acid residues that directly participate in catalysis (catalytic residues).

Regulatory Sites (if applicable)

Some enzymes have regulatory sites separate from the active site, where molecules such as inhibitors or activators can bind. These regulatory molecules can modulate the enzyme’s activity, inhibiting or enhancing its catalytic function.

Other structural components of enzymes

Some enzymes do not require chemical groups for activity except their amino acid residues. Others may require an additional chemical component called a cofactor—one or more inorganic ions, like Mg2, Mn2, Fe2, or Zn2, or a complex organic or metallo organic molecule called a coenzyme. Some enzymes require a coenzyme and one or more metal ions for activity. 

A prosthetic group is a coenzyme or metal ion that tightly or covalently binds to the enzyme protein. A holoenzyme is a complete, catalytically active enzyme with its bound coenzyme, with or without metal ions. The protein part of such an enzyme is an apoprotein or apoenzyme. 

Coenzymes can act as transient carriers of specific functional groups. Most are derived from vitamins and organic nutrients required in small amounts in the diet.

Classification and Nomenclature of Enzymes

The naming of many enzymes is done by adding the suffix “-ase” to their substrates’ name or a word or phrase that describes their activity. Thus, DNA polymerase catalyzes the polymerization of nucleotides to form DNA, and urease catalyzes the hydrolysis of urea. Before the specific reaction catalyzed was known, other enzymes were named for a broad function. For example, pepsin, from the Greek word pepsis, meaning “digestion,” was given to an enzyme known to act in the digestion of foods. 

Likewise, others were named for their source: trypsin, named in part from the Greek trying “to wear down,” was obtained by rubbing pancreatic tissue with glycerin. However, the same enzyme may have two or more names, or two different enzymes have the same name. Because of such ambiguities and the ever-increasing number of enzyme discoveries, international agreement led to the adoption of a system for naming and classifying enzymes. 

ClassActions
OxidoreductasesThese are responsible for the transfer of electrons (hydride ions or H atoms).
TransferasesThese aid in group transfer reactions.
HydrolasesThese are useful in hydrolysis reactions (transfer of functional groups to water).
LyasesThese help in the addition of groups to double bonds, or formation of double bonds by removal of groups.
IsomerasesThese enzymes assists in the transfer of groups within molecules to yield isomeric forms.
LigasesThese catalyzes the formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage.

Note: The function of most enzymes is the catalysis of the transfer of atoms, electrons, or functional groups. Therefore, they have different classifications, code numbers, and names assigned according to the type of transfer reaction, the donor group, and the group acceptor.

This system classifies enzymes into six classes based on the type of reaction they catalyze. These classes have their specific subclasses. Each enzyme has a four-part classification number and a systematic name identifying the reaction it catalyzes. 

For example, the formal systematic name of the enzyme catalyzing the reaction, ATP + D-glucose → ADP + D-glucose 6-phosphate, is ATP: glucose phosphotransferase. This denotes that the enzyme is the catalyst for transferring a phosphoryl group from ATP to glucose. 

The assigned Enzyme Commission number (E.C. number) for this enzyme is 2.7.1.1. The number 2 means the class name (transferase). Then, the number 2 denotes the subclass phosphotransferase. Similarly, the third number,1, denotes the acceptor as phosphotransferase having a hydroxyl group. Finally, the number, 1, means D-glucose is the acceptor for the phosphoryl group. For many enzymes, a trivial or common name is more commonly used. Hexokinase is the common name for this enzyme. 

IUBMB (International Union of Biochemistry and Molecular Biology) maintains a complete list describing the thousands of known enzymes. (https://iubmb.qmul.ac.uk/enzyme/rules.html). 

How do Enzymes Work?

The mechanism of enzyme action involves several key steps that occur at the molecular level. Enzymes facilitate biochemical reactions by lowering the energy needed for a reaction to proceed, thereby increasing the reaction rate. This process occurs through specific interactions between the enzyme, substrate, and other molecules. 

Substrate Binding

Enzyme substrate binding

The enzyme’s active site, which has a specific three-dimensional shape complementary to the substrate molecule, initially binds to the substrate. This binding can occur through a lock-and-key mechanism (where the substrate fits precisely into the active site) or an induced fit mechanism (where the active site reshapes slightly to accommodate the substrate).

Formation of the Enzyme-Substrate Complex

Once the substrate binds to the active site, it forms a temporary enzyme-substrate complex. This complex is stabilized by various interactions, such as van der Waals forces, hydrogen bonds, and electrostatic interactions between the enzyme and substrate.

Simple enzyme mechanism, showing the formation of the enzyme-substrate complex and the chemical step.

Catalysis

While bound to the enzyme, the substrate undergoes chemical transformations that lead to the formation of products. Enzymes catalyze these reactions by giving an alternative reaction pathway that requires less activation energy than the uncatalyzed reaction.

Active Site Residues 

Within the active site, specific amino acid residues play crucial roles in catalysis. These residues can act as acids or bases, nucleophiles, or participate in covalent bond formation with the substrate. Common catalytic residues include serine, cysteine, histidine, lysine, aspartate, and glutamate.

Transition State Stabilization

Enzymes stabilize the reaction’s transition state, the high-energy intermediate state that occurs during the conversion of substrate into product. By stabilizing the transition state, enzymes lower the activation barrier, making it easier for the reaction to proceed.

Product Release

After the catalytic reaction occurs, the enzyme releases the products chemically different from the original substrate. The enzyme is then free to bind to another substrate molecule and repeat the catalytic cycle.

Enzyme-Product Complex

In some cases, the enzyme may transiently form an enzyme-product complex after catalysis, where the product remains bound to the enzyme briefly before being released.

Regulation

Enzyme activity can be regulated through various mechanisms such as allosteric regulation, covalent modification, competitive and non-competitive inhibition, feedback inhibition, and enzyme induction or repression. These regulatory mechanisms ensure that enzyme activity is precise to the metabolic needs of the cell or organism.

Factors affecting Enzyme activity

Various factors can influence enzyme activity, impacting enzymatic reaction efficiency and rate. Understanding these factors is crucial for optimizing enzyme function in biological processes, industrial applications, and research settings. Here are the main factors affecting enzyme activity:

Temperature

Enzyme activity is susceptible to temperature changes. Generally, increasing temperature increases the rate of enzymatic reactions by providing more kinetic energy to molecules, which enhances their collision frequency and leads to more successful enzyme-substrate interactions. However, extremely high temperatures can denature enzymes, causing loss of their catalytic activity due to disruption of their three-dimensional structure. Each enzyme can operate at an optimal temperature range with maximum activity, known as the temperature optimum.

pH

The pH level of the environment influences enzyme activity by affecting the ionization state of amino acid residues at the active site. Enzymes have an optimal pH range at which they function most efficiently. Deviations from this pH range can alter the enzyme’s structure and charge distribution, reducing activity or denaturation. For example, pepsin, an enzyme in the stomach that digests proteins, works optimally in an acidic pH environment, while enzymes in the small intestine, such as pancreatic enzymes, function optimally in a slightly alkaline pH range.

Substrate Concentration 

Enzyme activity often depends on the concentration of substrate molecules available for binding to the enzyme’s active sites. As substrate concentration increases, enzyme activity increases because more enzyme-substrate complexes can form. However, at a certain point known as the saturation point, all enzyme active sites become occupied, and substrate concentration increases do not significantly increase the reaction rate. This point is essential in understanding enzyme kinetics and the concept of enzyme saturation.

Enzyme Concentration

The amount of enzyme in a reaction also affects enzyme activity. Generally, higher enzyme concentrations lead to faster reaction rates because more enzyme molecules are available to catalyze the conversion of substrates to products. However, like substrate concentration, there can be a saturation point where adding more enzymes does not increase the reaction rate if substrate concentration is limited.

Cofactors and Coenzymes

Many enzymes require specific cofactors or coenzymes for optimal activity. Cofactors can be metal ions (such as Mg2+, Zn2+, Fe2+) or organic molecules, while coenzymes are often vitamins or derivatives of vitamins. These cofactors and coenzymes assist enzymes in catalyzing reactions by participating in chemical reactions, transferring functional groups, or stabilizing reaction intermediates.

Inhibitors

Enzyme activity can be inhibited by various molecules known as inhibitors. Inhibitors are of two types: irreversible and reversible inhibitors. Reversible inhibitors include competitive inhibitors (compete with the substrate for the active site), non-competitive inhibitors (bind to an allosteric site and change the enzyme’s conformation), and uncompetitive inhibitors (bind to the enzyme-substrate complex). Irreversible inhibitors covalently bind to the enzyme, permanently inhibiting its activity.

Top: enzyme accelerates conversion of substrates to products. Middle: by binding to enzyme, inhibitor blocks binding of substrate. Bottom: by binding to enzyme, inhibitor disrups conversion of substrates to products. Binding sites in blue, substrates in black, inhibitors in green, allosteric site in light green.

Activators and Modulators

In contrast to inhibitors, activators and modulators can enhance enzyme activity. The molecules that bind to enzymes are activators and increase their catalytic activity. Allosteric activators bind to allosteric sites and induce conformational changes that enhance enzyme activity. Modulators can also alter enzyme activity by affecting conformation and activity through allosteric regulation.

Enzyme Structure and Conformational Changes

Enzyme structure and conformational dynamics play a crucial role in their activity. Changes in enzyme structure due to mutations, denaturation, or environmental factors can significantly affect enzyme activity and specificity.

Functions of Enzymes

Enzymes play vital roles in biochemical reactions and metabolic processes within living organisms. Their functions are diverse and essential for maintaining life processes. 

Catalysis

Enzymes act as biological catalysts, increasing biochemical reaction rates by using less activation energy. This catalytic activity allows cells to carry out metabolic processes efficiently, such as breaking down nutrients, synthesizing biomolecules, and producing energy.

Digestion

These play a crucial role in digestion by breaking down complex macromolecules into smaller, absorbable molecules. For example:

  • Amylase breaks down starch into glucose during carbohydrate digestion.
  • Proteases hydrolyze proteins into amino acids.
  • Lipases aid in the hydrolyzation of fats into fatty acids and glycerol.

Energy Production

Enzymes participate in energy production pathways like glycolysis, Krebs cycle, and oxidative phosphorylation. These pathways involve enzyme-catalyzed reactions that convert carbohydrates, fats, and proteins into energy-rich molecules such as ATP (adenosine triphosphate) that cells use for various cellular processes.

Synthesis of Biomolecules

Enzymes are involved in synthesizing biomolecules essential for cellular structure, function, and regulation. Examples include:

  • DNA polymerase and RNA polymerase catalyze the synthesis of DNA and RNA, respectively, during replication and transcription.
  • Although not enzymes, ribosomes facilitate protein synthesis (translation) by assembling amino acids into polypeptide chains based on mRNA instructions.

Detoxification

These in the liver and other organs participate in detoxification by metabolizing and eliminating harmful substances (such as drugs, toxins, and foreign compounds) from the body. These enzymes, such as cytochrome P450 enzymes, modify xenobiotics to make them more water-soluble for excretion.

Cell Signaling

Enzymes are involved in cell signaling pathways, where they catalyze reactions that regulate cellular responses to external stimuli, including hormones, neurotransmitters, and growth factors. Examples include protein kinases that phosphorylate proteins in signal transduction cascades and phosphatases that dephosphorylate them.

Immune Response

Enzymes are part of the immune system’s defense mechanisms. For instance, enzymes like lysozyme and proteases in tears, saliva, and mucus help protect against pathogens by breaking down their cell walls or proteins.

Repair and Maintenance

Enzymes participate in DNA repair mechanisms, ensuring genomic stability and integrity. Enzymes such as DNA repair polymerases, nucleases, and ligases recognize and correct DNA damage caused by various factors, including radiation, chemicals, and oxidative stress.

Regulation of Metabolic Pathways

These play a crucial role in regulating metabolic pathways by controlling the rates of specific reactions. Regulation can occur through feedback inhibition, allosteric regulation, covalent modification (such as phosphorylation), and gene expression changes affecting enzyme levels.

References

  1. Nelson, D., Lehninger, A., Cox, M., & Nelson, D. (2005). Lecture notebook for Lehninger principles of biochemistry, fourth edition (pp. 601-612). W.H. Freeman.
  2. odwell, V., Bender, D., Botham, K., Kennelly, P., & Weil, P. (2015). Harper’s illustrated biochemistry (30th ed., pp. 161-167). McGraw Hill.
Biochemistry, Molecular Biology

DNA Polymerase: Structure, Types, and Functions

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An enzyme, DNA polymerase, catalyzes the synthesis of new DNA molecules from deoxyribonucleotides (the building blocks of DNA). It is crucial in living organisms’ DNA replication, repair, and recombination processes. 

DNA polymerases attach new nucleotides to the 3′ end of a growing DNA strand by forming phosphodiester bonds between the new nucleotide and the existing DNA strand. This process ensures accurate copying of the genetic information stored in DNA during cell division and other cellular activities. 

DNA polymerases also possess proofreading capabilities to correct errors that may occur during DNA synthesis, contributing to the overall allegiance of DNA replication and maintenance of genetic integrity.

Structure of DNA Polymerase

DNA polymerases have a complex structure, allowing them to carry out their DNA replication and repair functions. The structure of DNA polymerase can vary slightly among different organisms and types of polymerases, but they generally share standard features. 

  1. Catalytic Core: DNA polymerase’s catalytic core contains the active site responsible for catalyzing the polymerization reaction. It includes domains involved in binding DNA and nucleotides and catalyzing the formation of phosphodiester bonds between nucleotides.
  2. Thumb Domain: This domain is involved in DNA binding and helps stabilize the interaction between DNA polymerase and the DNA template strand. It is called the “thumb” domain because of its structural resemblance to a thumb in some DNA polymerase structures.
  3. Palm Domain: The palm domain contains the active site of the polymerase enzyme, where nucleotide polymerization occurs. It also houses the catalytic residues, which are necessary to catalyze the addition of nucleotides to the growing DNA strand.
  4. Fingers Domain: The fingers domain is involved in the movement of the DNA and nucleotide substrates during the polymerization process. It undergoes conformational changes to allow the binding of incoming nucleotides and their incorporation into the growing DNA chain.
  5. Exonuclease Domain (Proofreading): Some DNA polymerases have an exonuclease domain responsible for proofreading the newly synthesized DNA strand. This domain can remove incorrectly incorporated nucleotides by excising them from the 3′ end of the growing DNA chain, improving the overall fidelity of DNA replication.
  6. Accessory Proteins: Besides the core catalytic domains, DNA polymerases often interact with accessory proteins that assist in various aspects of DNA replication, such as processivity, primer recognition, and coordination with other replication machinery components.

Overall, the structure of this is highly specialized to perform its essential functions in DNA replication and repair accurately and efficiently. Variations in structure and function exist among different DNA polymerase types, reflecting their diverse roles in maintaining genomic integrity across different organisms.

Types of DNA Polymerase

There are several types of polymerases involved in various DNA-related processes, including DNA replication, repair, and synthesis. The types of DNA polymerase vary among prokaryotes and Eukaryotes. 

Types of DNA Polymerase Found in Prokaryotes

Prokaryotic organisms, such as bacteria, have several DNA polymerases that play different roles in DNA replication, repair, and other cellular processes.

  1. DNA Polymerase I (Pol I):
    • Functions in DNA repair and gap filling during DNA synthesis.
    • It has 5′ to 3′ polymerase activity for DNA synthesis.
    • This type also possesses a 3′ to 5′ exonuclease (proofreading) activity, which helps correct errors during replication.
    • It also assists in removing RNA primers during Okazaki fragment maturation in DNA replication.
  2. DNA Polymerase II (Pol II):
    • She is involved in DNA repair processes, especially those that respond to DNA damage caused by environmental factors such as UV light.
    • It exhibits 5′ to 3′ polymerase activity and lacks 3′ to 5′ exonuclease activity.
  3. DNA Polymerase III (Pol III):
    • It is prokaryotes’ primary DNA replication enzyme, including bacteria like Escherichia coli (E. coli).
    • The highly processive enzyme is responsible for producing the leading as well as lagging strands during DNA replication.
    • They comprises of multiple subunits, including the core catalytic subunit responsible for DNA synthesis (Pol III core).
  4. DNA Polymerase IV (Pol IV) and DNA Polymerase V (Pol V):
    • These are specialized DNA polymerases involved in translesion DNA synthesis, particularly when regular DNA polymerases encounter DNA lesions or damage.
    • Pol IV and Pol V are error-prone and can replicate past damaged sites with reduced fidelity, allowing cells to tolerate DNA damage temporarily.
  5. DNA Polymerase VI (Pol VI):
    • Found in certain bacterial species.
    • Functions in translesion DNA synthesis and can bypass DNA lesions, similar to Pol IV and Pol V.

These DNA polymerases in prokaryotes work together in a coordinated manner during DNA replication, repair, and other DNA-related processes to ensure accurate DNA synthesis, maintain genomic stability, and respond to DNA damage. Each type has specific functions and characteristics that contribute to the fidelity and efficiency of DNA replication and repair in prokaryotic cells.

Types of DNA Polymerase Found in Eukaryotes

Eukaryotic organisms, like plants, animals, fungi, and protists, possess a variety of DNA polymerases that are required in different aspects of DNA replication, repair, and maintenance. 

  1. DNA Polymerase α (Pol α):
    • Involved in the initiation of DNA replication.
    • Synthesizes RNA-DNA primers on both leading and lagging strands during replication initiation.
    • Works in conjunction with other replication proteins to form the pre-replication complex.
  2. DNA Polymerase δ (Pol δ):
    • The main DNA replication enzyme is for the lagging strand during DNA replication.
    • It exhibits high processivity and fidelity.
    • Involved in synthesizing Okazaki fragments on the lagging strand.
  3. DNA Polymerase ε (Pol ε):
    • The main DNA replication enzyme is the leading strand during DNA replication.
    • Highly processive and accurate.
    • Works in coordination with other replication proteins to synthesize the leading strand continuously.
  4. DNA Polymerase β (Pol β):
    • Involved in base excision repair (BER) pathway.
    • This polymerase specializes in filling the gaps after damaged bases are removed during BER.
  5. DNA Polymerase γ (Pol γ):
    • It is present in mitochondria (mitochondrial DNA polymerase).
    • Responsible for replicating and maintaining the mitochondrial genome.
    • This type of polymerase plays a crucial role in mitochondrial DNA repair processes.
  6. DNA Polymerase η (Pol η):
    • Involved in translesion synthesis (TLS).
    • During DNA replication, specialized in bypassing UV-induced DNA lesions, such as thymine dimers.
  7. DNA Polymerase κ (Pol κ):
    • Involved in translesion synthesis (TLS).
    • Specialized in bypassing bulky DNA lesions, such as those caused by certain chemical agents.
  8. DNA Polymerase ι (Pol ι):
    • Involved in translesion synthesis (TLS).
    • Specialized in bypassing certain DNA lesions and insertions/deletions during DNA replication.
  9. DNA Polymerase ζ (Pol ζ):
    • Involved in translesion synthesis (TLS) and DNA damage tolerance.
    • Can replicate past DNA lesions with low fidelity, leading to mutagenesis under certain conditions.
  10. DNA Polymerase Rev1 (Pol Rev1):
    • Involved in translesion synthesis (TLS) and DNA damage tolerance.
    • It plays a role in generating mutagenic DNA synthesis during TLS.

These eukaryotic DNA polymerases have diverse functions and specialize in DNA replication and repair pathways. They contribute to maintaining genomic stability, accurately replicating DNA, and repairing damaged DNA to ensure the integrity of the genetic information in eukaryotic cells.

Functions

Diagram of DNA polymerase extending a DNA strand and proof-reading

DNA polymerase’s primary function is to act as a catalyst during the synthesis of new DNA strands by adding new nucleotides to the growing DNA chain during DNA replication, repair, and recombination processes. 

  1. DNA Replication: This enzyme plays a central role in DNA replication, where it copies the entire genome of a cell before cell division. During replication, DNA polymerase synthesizes new DNA strands complementary to the template strands. The leading strand synthesizes continuously in the 5′ to 3′ direction. However, the lagging strand synthesizes discontinuously in Okazaki fragments.
  2. Nucleotide Addition: It catalyzes the addition of deoxyribonucleotides (dNTPs) to the 3′ end of the growing DNA strand. It forms phosphodiester bonds between the incoming nucleotide and the last nucleotide of the ever-increasing strand, extending the DNA chain.
  3. Proofreading: Many DNA polymerases possess proofreading capabilities to maintain high fidelity during DNA synthesis. They have 3′ to 5′ exonuclease activity, allowing them to find and correct errors made during replication by removing incorrectly incorporated nucleotides from the 3′ end of the growing strand.
  4. Processivity: DNA polymerases are highly processive enzymes that can add multiple nucleotides sequentially without dissociating from the DNA template. This processivity ensures efficient DNA synthesis during replication and repair processes.
  5. DNA Repair: In addition to replication, DNA polymerases assist in DNA repair mechanisms. For example, during base excision repair, specialized DNA polymerases help fill the gaps left after the removal of damaged bases. Similarly, DNA polymerases are involved in nucleotide excision repair and mismatch repair pathways to correct various types of DNA damage and mismatches.
  6. Translesion Synthesis: Some DNA polymerases are specialized in bypassing DNA lesions or damage during replication. These polymerases, known as translesion polymerases, can replicate past damaged sites in the DNA template, albeit with reduced fidelity, to prevent replication stalling and maintain genome integrity.

References

  1. Nasheuer, Heinz & H.-P, & Pospiech, Helmut & Syvaoja, Juhani. (2006). DNA Polymerases.. 
  2. Steitz, T. A. (1999). DNA polymerases: Structural diversity and common mechanisms. Journal of Biological Chemistry, 274(25), 17395–17398. https://doi.org/10.1074/jbc.274.25.17395 
  3. Pospiech, H. and Syväoja, J.E. (2003) DNA Polymerase ε – More Than a Polymerase. TheScientificWorldJOURNAL 3, 87– 104. 
Biochemistry, Biotechnology, Molecular Biology

Immunofluorescence Assay: Principle, Steps, Types, and Uses

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Different diagnostic tests require other immunological processes to detect antibodies and antigens. Immunofluorescence assay is one of the most commonly used immunological tests. 

Immunofluorescence is the combination of the two words immuno and fluorescence. Immuno means immune or immunity; fluorescence implies fluorescent molecules that produce visible or invisible radiation. 

Immunofluorescence assay is a significant technique commonly used in immunology or molecular biology for detecting the presence and distribution of specific proteins or antigens. This process uses antibodies labeled with fluorescent molecules that bind to the target protein or antigen of interest. 

Principle of Immunofluorescence Assay

Immunofluorescence assay is based on specific antibodies for detecting and visualizing particular proteins or antigens in biological samples using fluorescence microscopy. 

The immunofluorescence assay principle relies on the antibodies’ specificity for their target proteins. This specificity helps selectively label and visualize the location and distribution of specific proteins within cells, tissues, or other biological samples. 

Basic mechanism of immunofluorescence

Immunofluorescence can be helpful in clinical diagnostics for detecting specific antigens or markers associated with diseases or conditions. 

The essential components of the immunofluorescence technique are antibodies and fluorescent labels.

  1. Antibodies: The antibodies are proteins the immune system produces that specifically bind to antigens. Likewise, the immune system recognizes antigens as foreign or non-self. In the immunofluorescence assay, specific antibodies target and attach to the protein or antigen of interest within a biological sample.  
  2. Fluorescent Labels: Fluorophores, or fluorescent labels, are attached to the antibodies. These labels emit fluorescent light when exposed to specific wavelengths of light. Each fluorophore emits light at a unique wavelength, which helps identify and differentiate different targets in the sample.   

General Steps of Immunofluorescence Assay

A general overview of the steps involved in an immunofluorescence experiment includes sample preparation, fixation, permeabilization, blocking, primary antibody incubation, washing, secondary antibody incubation, washing, mounting, and imaging. 

  1. Sample Preparation: In the case of cells, culture them on glass coverslips or chamber slides. While using tissues, prepare tissue sections by fixing, embedding, and sectioning the tissue. 
  2. Fixation: Chemical fixative (like formaldehyde) helps preserve the structure and immobilize the proteins while fixing the cells or tissues. 
  3. Permeabilization: Permeabilization is often necessary to enter antibodies inside the cells or tissues. This step involves treating the sample with the help of detergents or other agents. 
  4. Blocking: This step help to reduce the nonspecific binding of antibodies. Here, incubate the sample using a blocking solution like bovine serum albumin or normal serum. 
  5. Primary Antibody Incubation: Apply the primary antibody, specific to the target protein, to the sample. The antibody binds to the target protein within the sample. 
  6. Washing: The use of buffer solution helps in removing excess primary antibodies. 
  7. Secondary Antibody Incubation: Use an antibody conjugated to a fluorescent dye in this step. This secondary antibody recognizes the primary antibody and binds to it. 
  8. Washing: Like step 6, buffer solution helps remove excess secondary antibodies. 
  9. Mounting: Use a mounting medium with anti-fading agents for mounting the sample to preserve the fluorescence signal. 
  10. Imaging: The fluorescence microscope with appropriate filters examines the sample. This helps visualize the fluorescent signal emitted by the secondary antibody bound to the target protein. Different fluorophores can emit different colors of light, allowing for multicolor imaging. 

Types of Immunofluorescence Assay

There are different types of immunofluorescence assays with slight variations in the use and types of antibodies used. Common types of immunofluorescence assay are flow cytometry, immunohistochemistry, direct, indirect, multiplex, and double immunofluorescence assay.

  1. Direct Immunofluorescence or direct fluorescent antibody (DFA) test is a simple method requiring only primary antibody. The primary antibody conjugates to a fluorophore (fluorescent dye), which directly binds to the antigen of interest in the sample. 
  2. Indirect Immunofluorescence or indirect fluorescent antibody (IFA) test is the most common immunofluorescence technique. Firstly, application of a primary antibody specific to the targeted antigen occurs. Then, there is the use of a secondary antibody labeled with a fluorescent dye particular to the primary antibody. Here, the secondary antibody binds to primary antibodies. The added step makes this process more sensitive. 
  3. Immunohistochemistry (IHC) combines immunofluorescence with tissue sectioning. Here, treatment of tissue sections with antibodies labeled with fluorescent dyes occurs. It helps in visualizing the distribution and localization of specific antigens within tissues.
  4. Flow Cytometry combines immunofluorescence with analysis of individual cells in a liquid stream. Each cell is labeled with fluorescently tagged antibodies and passed through a flow cytometer. The flow cytometer measures the fluorescence intensity of each cell. It is applicable in cell sorting, cell phenotyping, and quantifying surface markers on cells.   
  5. Immunocytochemistry helps to detect specific proteins within cultured cells. Cells are fixed and then treated with labeled antibodies for visualization. 
  6. Western Blotting is applicable in detecting and quantifying proteins. Here, gel electrophoresis combines with immunofluorescence, where protein separation occurs by size in the gel. Researchers then transfers protein to a membrane and probed with antibodies labeled with fluorescent dyes. 
  7. Multiplex immunofluorescence helps detect multiple antigens simultaneously within a single sample. Here, different primary antibodies are labeled with fluorophores, which helps visualize multiple antigens or markers in a single experiment. 
  8. Double immunofluorescence detects two different antigens within the same sample. Here, there is use of two primary antibodies from other species. Recognition of each occurs by a different secondary antibody labeled with distinct fluorophores. This method helps in studying co-localization or interactions between two proteins. 

Benefits

Immunofluorescence assay is useful widely in biomedical research and diagnostics because of several key benefits. The benefits are briefly explained below:

  1. Immunofluorescence is highly specific in detecting target proteins due to antibodies binding specifically to the interested antigen. 
  2. This method is susceptible because fluorescent dyes amplify the signal, helping detect protein even in low-abundance samples.
  3. It helps visualize the distribution and location of proteins within cells and tissues. It also provides insights into the spatial organization of cellular structures, including subcellular compartments, cellular membranes, and organelles.  
  4. Immunofluorescence helps in studying protein-protein interactions and co-localization within the same cellular compartment. 
  5. A type of immunofluorescence helps in detecting multiple proteins and antigens within a single sample. 
  6. This technique applies to various biological samples, including cells, tissue, and whole organisms. It is also helpful in cell biology, neuroscience, immunology, pathology, and diagnostics. 
  7. This method also helps quantify proteins or antigens with appropriate imaging and analysis tools.
  8. Using fluorescence microscopy helps in obtaining detailed images of cellular structures and proteins. 
  9. Likewise, this technique is compatible with other techniques like electron microscopy for visualizing the ultrastructure of cells and tissues. 
  10. The signals from fluorescence are stable. Also, the signals can be preserved for imaging and analysis over time, which helps reanalyze the sample. 

Limitations

Although immunofluorescence is highly beneficial, it has some limitations. Researchers must consider these limitations and challenges before selecting this method. Some of the critical limitations of immunofluorescence assays are:

  1. Sometimes, false positive results may occur due to non-specific binding of antibodies to unrelated molecules. It may also yield false negative results due to low antigen expression or epitope masking. 
  2. Cross-reactions with closely related antigens may occur. So, antibody validation and specificity testing are essential to address this issue. 
  3. Selecting appropriate and quality antibodies is a challenging and vital step in immunofluorescence assay. 
  4. The background signal from the sample can interfere with the specific signal, so proper controls and background subtraction methods are necessary to address the issue. 
  5. While performing tissue-based immunofluorescence, fixation, and permeabilization methods can impact the results because fixation methods can alter protein structure, affecting antibody binding. 
  6. Fluorescent dyes can be sensitive to photobleaching, reducing the intensity of the fluorescence signal over time. Hence, researchers must minimize exposure to intense light.
  7. Accurate quantification of immunofluorescence signals can be challenging due to variations in fluorescence intensity, background noise, and the dynamic range of detection. 
  8. This method can be expensive and time-consuming. Also, this method requires expertise.

References

  1. Im, K., Mareninov, S., Diaz, M. F. P., & Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. Methods in molecular biology (Clifton, N.J.), 1897, 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 
  2. The Principle of Immunofluorescence Assays. Retrieved from https://ibidi.com/content/364-the-principle-of-immunofluorescence-assays  
Biochemical Tests, Biochemistry

Mixed Acid Fermentation: Types and Products

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Fermentation is an anaerobic process of breaking down molecules like glucose and other carbohydrates. The fermentation process is usually helpful in alcohol production. Bacteria follow different fermentation pathways.

Among them, mixed acid fermentation is a characteristic feature of the family Enterobacteriaceae, especially the genera Citrobacter, Proteus, Shigella, Salmonella, Escherichia, Aeromonas, Yersinia, Vibrio, and some species of Aeromonas. Some anaerobic fungi also follow this pathway.

These microorganisms ferment monosaccharides, disaccharides, polyalcohol, and frequently polysaccharides. The glycolytic pathway of this type of fermentation produces lactic acid, succinic acid, formic acids, acetic acids, and ethanol.

Reactions Involved in Mixed Acid Fermentation

In mixed acid fermentation reactions, two stages are present. The first stage of mixed acid fermentation is glycolysis, converting glucose to pyruvate. Here two NADH molecules are produced.

The second stage of mixed acid fermentation follows the following reactions the conversion of the pyruvate produced after glycolysis to one or more end products. The two NADH molecules produced in the first stage are reoxidized to NAD+. The reactions in the second stage are discussed below.

Mixed acid fermentation in E coli

Lactate Production

The enzyme lactate dehydrogenase catalyzes the formation of lactate/lactic acid. Here, glycolysis generates two molecules of pyruvate. Each molecule converts to lactate in the presence of a NADH+H+ molecule.

The overall reaction of lactate production is as follows:

Pyruvate → Lactate; in presence of lactate dehydrogenase and NADH+H+, which converts to NAD+

Acetate Production

In this reaction, pyruvate converts to acetyl CoA with the enzyme pyruvate dehydrogenase and NADH catalysts. The acetyl CoA now converts to acetate, which produces ATP by substrate-level phosphorylation. The conversion of acetyl CoA to acetate is a two-step process requiring two separate enzymes; phosphate acetyltransferase and acetate kinase.

The overall reaction of this reaction is as follows;

  1. Pyruvate → Acetyl CoA in the presence of pyruvate dehydrogenase
  2. Acetyl CoA + Phosphate → Acetyl phosphate + CoA in the presence of phosphate acetyltransferase.
  3. Acetyl phosphate + ADP → Acetate + ATP. 

Ethanol Production

The reduction of Acetyl CoA with the help of NADH forms the third end product of mixed acid fermentation; ethanol. This ethanol production is a two-step reaction and requires the enzyme alcohol dehydrogenase.

The overall reaction of this step of fermentation is;

  1. Acetyl CoA + NADH +H+ → Acetaldehyde + NAD+ + CoA
  2. Acetaldehyde + NADH + H+ →  Ethanol + NAD+ 

Formate Production

The cleavage of pyruvate helps in the production of Formate. The enzyme pyruvate-formate-lyase catalyzes this production reaction. The enzyme pyruvate-formate-lyase also plays an essential role in regulating anaerobic fermentation in Enterobacteriaceae.

The overall reaction of the formate production is as follows;

Pyruvate + CoA  → Acetyl CoA + Formate; catalyzed by pyruvate-formate-lyase. 

Succinate Production

The production of succinate is a multi-step process. The glycolytic pathway intermediate, phosphoenol pyruvate, is the first substrate for carboxylation to form oxaloacetate, with the enzyme phosphoenol pyruvate carboxylase catalyzing this step.

In the second step, the oxaloacetate converts to malate in the presence of malate dehydrogenase. The third step is the formation of fumarate from the dehydration of malate in the fact of fumarate hydratase.

Phosphoenol pyruvate + HCO3 → Oxaloacetate +Phosphate

Oxaloacetate + NADH + H+ → Malate + NAD+ 

Malate → Fumarate + H2O

The final step is succinate production by reducing formate catalyzed by the enzyme fumarate reductase.

Fumarate + NADH + NAD+ →  Succinate + NAD+

The reduction is the anaerobic respiration reaction that uses electrons in NADH dehydrogenase and the electron transport chain. ATP is produced using electrochemical balance and ATP synthetase—this step of fermentation produces ATP, not through substrate-level phosphorylation.

Hydrogen and Carbon Dioxide Production

The enzyme formate hydrogen lyase catalyzes the conversion of formate to hydrogen carbon dioxide gas. The production of these gases helps in preventing acidic condition inside the cells.

End Products of Mixed Acid Fermentation

The end products of mixed acid fermentation are as follows:

  1. Lactate
  2. Acetate
  3. Formate
  4. Succinate
  5. Ethanol

The concentration of these end products of fermentation vary depending on the environment and microorganism but it may be at the 4:1 ratio of neutral to acid products. Hydrogen and carbon dioxide is formed in bacteria with formate hydrogen lyase complex.

Application of Mixed Acid Fermentation

Mixed Acid fermentation is applicable in various fields of science, especially for producing many helpful end products. The applications of mixed acid fermentation are as follows:

  1. The use of single bacteria can help produce various products in biotechnology and the food industry.
  2. Likewise, many different strains of bacteria have been metabolically engineered in the laboratory to increase the yield of the specific end product.
  3. This fermentation method is applied to identify bacteria in the laboratory. Methyl red test is standard for detecting the bacteria following the mixed acid fermentation reaction. Here, the test solution turns red if the pH drops below 4.4 in the presence of those microorganisms that follows the mixed acid fermentation pathway.

References

  1. Thakker, C., Martínez, I., San, K. Y., & Bennett, G. N. (2012). Succinate production in Escherichia coli. Biotechnology journal, 7(2), 213–224. https://doi.org/10.1002/biot.201100061
  2. Vuoristo, K. S., Mars, A. E., Sangra, J. V., Springer, J., Eggink, G., Sanders, J. P., & Weusthuis, R. A. (2015). Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate. AMB Express, 5(1), 61. https://doi.org/10.1186/s13568-015-0147-y
  3. Böck, A. (2009). Fermentation. Encyclopedia of Microbiology, 132–144. https://doi.org/10.1016/b978-012373944-5.00074-2
Biochemistry

Cori Cycle: Steps, Regulation, and Importance

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Different cells in the human body need to release energy during high periods of energy demand, like during intense exercise. The Cori cycle is a metabolic pathway involving the interconversion of glucose and lactate between the muscles and the liver.

The Cori cycle is called lactic acid shuttle or lactic acid cycle. Cori Cycle is named after the husband and wife scientists duo Gertrude (Gerty) and Carl Cori, who introduced and descirbed this cycle from 1925 to 1950 AD. However, they jointly recieved the novel prize in physiology and medicine in 1947 for the discovery of this cycle. 

The cycle completes in a five-step process where four ATP molecules are used. The five steps involve; Lactate production, Transport of lactate to the liver, Conversion of lactate to glucose, The release of glucose in the bloodstream, and Uptake of glucose by other cells. 

Steps of Cori Cycle

This metabolic pathway requires three different types of human cells; liver, blood, and muscle or other high energy-requiring cells. This cycle is vital for maintaining glucose levels during high energy demand periods like intense exercise. 

The Cori cycle links anaerobic glycolysis in muscle tissue to gluconeogenesis in the liver.

As discussed earlier, the Cori cycle occurs in five steps; lactic acid production, transportation of lactate to the liver, glucose production, the release of glucose in the blood, and glucose uptake by required cells. 

  1. Lactate production: Glycogen stored in the muscle cells converts to glucose via glycogenolysis. During high energy demand, especially in muscle cells, the pyruvate produced after glycolysis of glucose follows the anaerobic pathway to produce lactic acid or lactate. The anaerobic fate of pyruvate occurs due to a lack of oxygen in these cells. So, this cycle is also termed anaerobic glycolysis for lactic acid production. Here, the conversion of glucose to pyruvate produces two ATP molecules.  
  2. Transport of lactate to the liver: The lactate produced cannot be utilized by muscle cells. Liver cells can only use lactate. So it is transported to liver cells via the bloodstream. 
  3. Gluconeogenesis: Gluconeogenesis is a metabolic pathway that helps produce glucose from non-carbohydrate compounds in the liver cells. During the lactate cycle, lactic acid converts into glucose. It is a complex procedure requiring multiple enzymes. It requires six ATP molecules. 
  4. Release of glucose in the blood: The newly formed glucose is released into the bloodstream. This release helps maintain the blood glucose level during high-intensity exercise periods. Glucose is a fuel source for tissues, including the brain, RBCs, and muscles.  
  5. Glucose uptake by muscles and other tissues: The glucose in the bloodstream is uptaken by cells like muscles, the brain, and other tissues. Here the glucose converts into pyruvate by glycolysis, which follows the aerobic fate to produce carbon dioxide. The CO2 is then released outside the body via the lungs.  

Energy Calculation

The Cori Cycle

The energy calculation during the Cori cycle is as follows:

Glucose/Glycogen → Pyruvate; produces 2 ATP molecules

Lactate → Glucose; requires 6 ATP molecules

So, a total of 4 ATP molecules is used in this cycle. 

Although it uses four molecules of ATP, it is an essential cycle for energy production. This high amount of energy production is because, after the glucose uptake by different cells, the glucose enters the TCA cycle, producing almost 10 ATPs per acetyl CoA molecule.

Read more about TCA cycle here.  

Regulation of Cori Cycle

As discussed earlier, lactic acid cycle is an essential process in the body that helps control blood glucose levels and provides energy during an intense situation. So, this cycle must be tightly regulated by internal as well as external factors. Numerous factors regulate the Cori cycle. External factors include exercise intensity and nutrition intake, and internal factors include hormonal control, oxygen, and glucose availability.

  1. Hormonal regulation: The hormones insulin, glucagon, and adrenalin help regulate the Cori cycle. Adrenalin is a stress hormone that promotes the release of glucose in the bloodstream from liver cells. Glucagon prevents the blood glucose level from dropping lower than average blood sugar level, so in the case of the Cori cycle, glucagon also promotes the release of glucose in the blood. Insulin helps uptake glucose from the blood into the targeted cell.   
  2. Regulation due to exercise intensity: The higher the power, the more energy is required. So, the higher the exercise intensity, the higher the number of Cori cycles. 
  3. Nutritional intake: High carbohydrate diet enhances/promotes the steps of the Cori cycle. Likewise, the higher the amount of lipid and protein in the diet, the less chance of Cori cycle occurrence. 
  4. Availability of oxygen: If oxygen is unavailable in muscle cells, it triggers the formation of lactate in the cell. Lactate act as the substrate for the Cori cycle. Once oxygen is available, the Cori cycle is halted.  
  5. Glucose availability: Once the glucose level in the blood drops, the Cori cycle activates, which helps increase the blood glucose level. Whereas, once the blood glucose level increases and uptake by the required cells is complete, the Cori cycle stops.  

Importance of Lactic Acid Cycle

The lactic acid cycle is a crucial mechanism that helps adapt the body to various energy demands. It ensures a steady supply of glucose in cells even during a temporary shortage of oxygen. It is the most essential role of the lactic acid cycle. Let us discuss the importance of the Cori cycle in the human body: 

  1. Prevent acidosis in muscles: Acidosis is the accumulation of acid in cells. In the case of muscle cells, lactate or lactic acid accumulates in the absence of oxygen, causing cramps, nausea, and weakness. It usually can happen during exercise, and activation of the Cori cycle helps prevent acidosis.   
  2. Increase exercise intensity: Exercise demands a high amount of energy without oxygen. The Cori cycle can help increase glucose levels in the blood by utilizing the lactate from muscles. The glucose helps increase power which helps in increasing the intensity of exercise. 
  3. Maintain glucose level and energy during stressful times: Cori cycle helps maintain the level of glucose and vitality during stressful times, not only exercise but also mental stress. So, it is also an essential process during flight or fight response. 

Disorders Related to Cori Cycle 

The disruption and absence of the Cori cycle in humans can cause many complications, like Cori’s disease, lactic acidosis, McCardle’s disease, and hypoglycemia during exercise. 

  1. Cori’s disease: Glycogen storage disease IIi (GSD-IIi) or Cori/Forbes disease is a genetic condition that stops the breakdown of glycogen to glucose. The breakdown is halted due to the absence of enzymes required for glycogenolysis. This condition causes muscle weakness, hypertrophy of the liver (enlargement of the liver), and delayed growth in children. 
  2. Exercise-induced hypoglycemia: Lack of the Cori cycle can cause lead to exercise-induced hypoglycemia (EIH). EIH can cause dizziness and weakness while doing exercise. Here the blood glucose level decreases significantly. 
  3. McArdle’s disease: It is also a genetic disorder where an individual lacks the enzyme to break down glucose. People with this disease can experience fatigue, muscle cramps, and weakness during physical exercise. 
  4. Lactic Acidosis: Lactic acidosis is a disorder where accumulation of lactic acid occurs in the skeletal muscle cells. It can lead to extreme fatigue, body weakness, abdominal discomfort, and headache. 

References

  1. Matthews, C. K., van Holde, K.E., and Ahern, K. G., Biochemistry, 3rd Ed., Addison Wesley Longman, 2000
  2. National Center for Biotechnology Information (2023). PubChem Pathway Summary for Pathway WP1946, Cori cycle, Source: WikiPathways. Retrieved August 9, 2023 from https://pubchem.ncbi.nlm.nih.gov/pathway/WikiPathways:WP1946.
  3. Nelson, D. L., Cox, M. M., & Lehninger, A. L. (2005). Hormonal Regulation and Integration of Mammalian Metabolism. In Principles of biochemistry (3rd ed., pp. 898–899). essay, Freeman.
Biochemistry, General Microbiology

Beta (𝛃) Oxidation: The Body’s Way of Utilizing Fats

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The fats in the body are triacylglycerols, cholesterol, or long chains of fatty acids. The fatty acids, a biomolecule, are converted into acetyl-CoA by the method known as beta (𝛃)oxidation. 

The acetyl CoA enters the central energy-yielding pathway in many organisms and tissues. Likewise, the electrons removed during 𝛃-oxidation pass through the respiratory chain. Acetyl CoA can also convert to ketone bodies and act as water-soluble fuels for the brain and other tissues. 

Beta (𝛃) oxidation is a repetitive four-step metabolic process by which long-chain fatty acids convert into two-chain carbon compounds (acetyl CoA). The fatty acids are insoluble in water and chemically inert, making these perfect storage fuels. 

These properties make their catabolism challenging. So, the complete oxidation of fatty acids to CO2 and H2O has primarily three stages; 𝛃-oxidation, oxidation of acetyl CoA in the citric acid cycle, and transfer of electrons to the mitochondrial respiratory chain. 

The cells gain fatty acids from three sources; from the diet, fats stored as lipid droplets, and fats synthesized in one part and exported to another. Some species use all three sources and others may use one or two sources. Vertebrates use all three sources, whereas protists obtain fat from their diet only.

Transportation of Fatty Acids

Even though the fats come from different sources, these should enter the mitochondria of the cells before 𝛽-oxidation. The method of transfer of fats depends on the source. Like small intestine absorbs dietary fats. Through blood, it reaches the liver, where it’s catabolism occurs. The fats absorbed from the intestine can also be stored as fat droplets in the adipose and muscle tissues.    

The enzymes for the oxidation of fatty acids reside inside the mitochondrial matrix. The fatty acids with 12 or fewer carbon molecules can easily enter the mitochondria without membrane transporters. The free fatty acids (FFAs) with 14 or more carbon molecules cannot enter the mitochondrial membranes. So these must undergo the three-step enzymatic reactions of the carnitine shuttle. 

The family of isozymes present in the outer mitochondrial membrane, the acyl-CoA synthetases, catalyzes the first reaction of the shuttle. Thus, forming a fatty acyl-CoA. The thioester linkage between the thiol group of coenzyme A and the carboxyl group of fatty acid forms the fatty acyl-CoA. Here, the cleavage of ATP to AMP and PPi is the energy source for this reaction. The reaction occurs in two steps. The first step is formation of intermediate product, fatty acyl-adenylate, bounded by enzyme. The second step is the formation of fatty acyl CoA. The step is also called activation of fatty acids. 

Reaction Involved:

Fatty acid + ATP ⇋ Fatty acyl–adenylate + CoA ⇋ Fatty acyl CoA + AMP + PPi; in presence of acyl-CoA synthetase. △G° = -34 kJ/mol

Like acetyl CoA, fatty acyl CoA is a high-energy compound because the hydrolysis to FFA and CoA has a significant, negative standard free-energy change, i.e.,△G ≅ 31 kJ/mol. The hydrolysis of two phosphate molecules of ATP makes this reaction favorable. In the outer membrane of the mitochondria (face towards the inner membrane), fatty acyl-CoA oxidizes to produce ATP. The ATP returns to the cytosol facing the side of the outer membrane and reused.    

In the shuttle’s second reaction, the carnitine hydroxyl group transiently attaches to the fatty acid that will be entering inside the mitochondria. This reaction forms fatty acyl carnitine under the catalyst carnitine acyltransferase I. The CoA is removed either in the outer membrane or intermembrane space. However, the exact place of CoA removal is not discovered. The fatty acyl-carnitine ester enters the mitochondrial matrix by facilitated diffusion through the acyl-carnitine/carnitine transporter of the inner mitochondrial membrane.    

The carnitine shuttle’s final reaction is to transfer the fatty acyl group enzymatically from carnitine to mitochondrial CoA by carnitine acyltransferase II. The enzyme is located on the inner face of the inner mitochondrial membrane for releasing fatty acyl-CoA and free carnitine into the matrix. The free carnitine reenters the intermembrane space through the acyl-carnitine transporter. 

Steps of Beta (𝛃) Oxidation

The first step of mitochondrial oxidation of fatty acids is the oxidative removal of successive two-carbon units in the form of acetyl CoA from the carboxyl end of the fatty acyl chain. For example, for 16-carbon palmitic acid undergoes seven passes and each pass releases two carbons as acetyl CoA forming 8 acetyl CoA.  

𝛃-oxidation of saturated even numbered fatty acids has four basic steps. For unsaturated even numbered fatty acids there is three additional steps. Finally, four additional steps are required for odd numbered fatty acids.  

𝛃-Oxidation of Even Saturated Fatty Acids

The beta-oxidation of even saturated fatty acids has four basic steps. The steps repeat until the entire fatty acid completely oxidizes. For example, myristic acid has 14 carbon, and the four basic steps repeat six times to produce seven acetyls CoA. 

  1. The first step of β-oxidation is the dehydrogenation of fatty acyl-CoA to produce a double bond between ɑ and β carbon atoms (C-2 ad C-3) that yields trans-2-enoyl-CoA. Three isozymes of acyl-CoA dehydrogenase catalyzes this reaction. Each enzyme is specific for a range of fatty-acyl chain lengths; very long chain (12-18), medium chain (4-14), and short-chain acyl-CoA dehydrogenase (4-8). A molecule of FAD reduces to FADH2 by the electrons produced after the addition of the double bond. 
  2. Then, in the second step of the β-oxidation, addition of water to the double bond of the trans-2-enoyl-CoA occurs. It forms the L stereoisomer of β-hydroxy acyl-CoA, catalyzed by enoyl-CoA hydratase. 
  3. After that, the third step is the dehydrogenation of L-hydroxy acyl-CoA to form β-ketoacyl-CoA, with the help of catalyst β-hydroxy acyl-CoA dehydrogenase. NAD is the electron acceptor for this step which reduces to NADH + H+. 
  4. Acyl-CoA acetyltransferase (thiolase) catalyzes the last step of β-oxidation where the carboxyl-terminal of the β-ketoacyl-CoA splits to form acetyl-CoA. This step requires a molecule of free coenzyme A. The other byproduct is the coenzyme A thioester of the fatty acid with two fewer carbon atoms. The reaction is called thiolysis because of the removal of the thiol group of coenzyme A.  
Beta oxidation of palmitic acid

For fatty acyl chains of 12 or more carbons, the multienzyme complex, the trifunctional protein (TFP), catalyzes the reactions. The TFP is a hetero-octamer 44 subunit. After the long-chain fatty acids shorten to 12 or fewer carbons, four soluble enzymes in the matrix catalyzes the further oxidations.

𝛃-Oxidation of Even Unsaturated Fatty Acids

Most of the fatty acids in phospholipids and triacylglycerols of animals are usually unsaturated, i.e., with one or more double bonds. Enoyl-CoA hydratase cannot act upon the double bonds. β-oxidation of common unsaturated fatty acids requires two added enzymes; isomerase and reductase. 

Isomerase changes the position of the double bond to the right position, i.e., from cis to trans in monosaturated fatty acids. Reductase and isomerase work together to change the position of two cis double bonds in polyunsaturated fatty acids.

Beta oxidation of linolenic acid

To understand the concept of 𝛃-oxidation of even monounsaturated fatty acids, let us take an example of oleate, an 18-carbon monounsaturated fatty acid with a cis double bond between C-9 and C-10. Its oxidation includes the following steps:

  1. The oleate converts to oleoyl-CoA occurs like typical saturated fatty acids and enters the mitochondrial matrix through the carnitine shuttle. Oleoyl-CoA undergoes three cycles of normal 𝛃-oxidation forming three molecules of acetyl-CoA and the CoA ester of a 12-carbon unsaturated fatty acid, cis-3-dodecenoyl CoA. 
  2. The auxiliary enzyme 3,2-enoyl-CoA isomerase changes the cis-3-dodecenoyl-COA to the trans-2-enoyl-CoA which is the substrate for the enzyme enoyl CoA hydratase and converts it into the L-𝛃-hydroxyacyl-CoA and releasing a molecule of acetyl CoA. 
  3. Now, the other enzymes oxidizes the intermediate product in four cycles of beta-oxidation to yield five more acetyls CoA. Hence, a total of nine acetyls CoA forms under the beta-oxidation of oleate.  

𝛃-Oxidation of Odd Fatty Acids

Although most of the fatty acids utilized by the body are even-number, odd-number fatty acids are also reasonably common in many plants and marine organisms. The activation of odd fatty acids for oxidation is as same as even-number fatty acids, i.e., beginning at the carboxyl end of the chain. 

Beta oxidation of odd-numbered fatty acid

The substrate for the final pass of ꞵ-oxidation is a 5-fatty acyl-CoA, which oxidizes to form acetyl-CoA and propionyl-CoA. The acetyl-CoA enters the citric cycle, whereas propionyl-CoA enters a different pathway. 

In the first step, propionyl-CoA carboxylated to form a D-stereoisomer of methyl malonyl-COA in the presence of propionyl-CoA carboxylase, ATP, and HCO3–. Cleaving of ATP to ADP and Pi provides energy for this reaction. The methyl malonyl-CoA epimerase transforms D-methyl malonyl-CoA into L-stereoisomer (L-methyl malonyl-CoA). methyl malonyl-CoA mutase and coenzyme B12 catalyzes the rearrangement of L-methyl malonyl-CoA to form succinyl-CoA. The succinyl-CoA enters the citric cycle. 

Energy Yield During Beta Oxidation

The energy-yielding steps in beta-oxidation are:

  1. The first step is where fatty acyl-CoA converts into tans-2-Enoyl-CoA, and forming an FADH2 molecule. For the “n” number of cycles, “n” FADH2 forms. Each FADH2 produces 1.5 ATP after oxidative phosphorylation.
  2. The third step is where ꞵ-hydroxy acyl-CoA converts into ꞵ-ketoacyl-CoA, a molecule of NADH, i.e.,producing “n” NADH after the “n” number of the ꞵ-oxidation cycle. From a single NADH, 2.5 ATP produces after oxidative phosphorylation. 
  3. Each cycle of ꞵ-oxidation produces a molecule of acetyl CoA, so the “n” cycle of ꞵ-oxidation produces “n+1” acetyl CoA. Each molecule of acetyl-CoA produces 10 ATP after Kreb’s cycle. 

So for example, beta oxidation of palmitoyl-CoA produces 7 FADH2, 7 NADH, and eight acetyl-CoA, which enters the citric acid cycle and oxidative phosphorylation to produce 108 ATP molecules (10.5 from FADH2, 17.5 from NADH, and 80 from acetyl CoA). 

Two molecules of ATP are used during activation, so 106 ATP is generated after the oxidation of palmitate. 

Regulation of Beta 𝛃 Oxidation

The ꞵ-oxidation of fatty acid is highly regulated as it is a necessary fuel, and oxidation must only begin in dire situations. 

The three-step process (carnitine shuttle) that transfers fatty acids from cytosol to mitochondrial matrix is rate limiting for ꞵ-oxidation and a key regulation point. It is because once the fatty acyl CoA group enters the mitochondria, it will be converted into acetyl CoA. The following is the way this shuttle is regulated:

  1. The first intermediate byproduct of cytosolic biosynthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, increases in concentration whenever there is a good supply of carbohydrates. This byproduct inhibits acyltransferase I inhibiting the ꞵ-oxidation of fatty acids in the liver.
  2. When NADH/NAD ratio is high, inhibition of ꞵ-hydroxy acyl-CoA dehydrogenase occurs. 
  3. High concentrations of acetyl CoA inhibit thiolase.  
  4. Insulin also inhibits ꞵ-oxidation by dephosphorylating hormone-sensitive lipase, which inhibits the release of fatty acids from adipose tissues.   

Activating ꞵ-oxidation occurs due to the epinephrine activating a cAMP-dependent protein kinase. The cAMP-dependent protein kinase phosphorylates and activates hormone-sensitive lipase. Hormone-sensitive lipase releases fatty acids and glycerol from adipose tissue for ꞵ-oxidation.   

Importance of 𝛃-Oxidation

Ꞵ-oxidation is an essential metabolic process because it yields energy during exercising (in humans) or during hibernation in hibernating animals like bears. Other importance of ꞵ-oxidation include:

  1. It helps control blood glucose levels, i.e., people with genetic defects have reported low blood glucose levels (hypoglycemia).  
  2. The non-alcoholic fatty liver syndrome occurs due to impaired beta-oxidation.    

References

  1. Nelson, D., Lehninger, A., Cox, M., & Nelson, D. (2005). Lecture notebook for Lehninger principles of biochemistry, fourth edition (pp. 631-643). W.H. Freeman.
  2. Talley JT, Mohiuddin SS. Biochemistry, Fatty Acid Oxidation. [Updated 2023 Jan 16]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK556002/ 
Biochemistry, General Microbiology

Bial’s Test: Principle, Procedure, and Application

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Carbohydrates are one of the three essential nutrients; the other two are proteins and fats. Molisch’s, Benedict’s, Fehling’s, Tollen’s, Iodine and Bial’s test are the common tests for determining the presence of carbohydrates in the analytes.

Bial’s test helps separate pentose and pentose-derived compounds (pentosans) from other carbohydrates. Pentoses are simple sugars with five carbon molecules as the backbone. The most common examples of pentose and its derivatives are xylose, ribose, arabinose, hemicellulose, DNA (deoxyribonucleic acid), and RNA (ribonucleic acid).

Principle of Bial’s Test

Bial’s test aims to determine the presence of pentose and pentose-derived compounds in analytes. It also helps in the differentiation of pentose monosaccharides from other carbohydrates.

This test depends on the principle that when reagent reacts with a sample containing pentose or derivatives of pentose, it gives a blue-green complex. Here, derivatives of pentose degrades to pentose. Then, the dehydration of pentose occurs due to concentrated HCl (hydrochloric acid) in the Bial’s reagent forms furfural.

Pentose/pentose derived compounds + H+ (from concentrated HCl) → Furfural 

Furfural + FeCl3 + Orcinol → Blue-green complex

The furfural reacts with orcinol in the presence of ferric ions to form a blue-green complex. The hexose monosaccharides in the sample react with conc. HCl forms a 5-hydroxymethyl furfural that reacts with orcinol in the presence of ferric ions, giving a muddy-brown complex.

Hexose/hexose derived compounds + H+ → 5-hydroxymethyl furfural 

5-hydroxymethyl furfural + FeCl3 + orcinol → Muddy-brown complex

Materials Required

There are different materials required for performing this test. Reagents and equipment needed for the tests are Bial’s reagent, test tubes, dropper, and water bath.

Reagent Required

Bial’s reagent, distilled water, xylose solution, and sample suspension are required reagents for performing this experiment. The reagent consists of orcinol, concentrated hydrochloric acid, and ferric chloride. The reagent must be fresh, i.e., prepared before performing the test.

Preparation of Bial’s reagent

  1. Prepare 10% ferric chloride by dissolving 10 g of FeCl3 in 100 ml distilled water.
  2. Dissolve 1.5 g of orcinol in 500 ml of concentrated HCl.
  3. Then, add 1 ml or 20 drops of the 10% FeCl3 solution.
  4. Finally, store the sample in a dark brown bottle before use. The reagent must be used within a couple of hours.

Equipment Required

The equipment used and their purpose in this test are as follows:

  1. Test tube: It is a cylindrical tube made of glass. Its use is to mix the sample solution with the Bial’s reagent.
  2. Water Bath: It is the laboratory equipment that consists of hot water. It is used for heating the test tubes over constant temperature.
  3. Reagent bottle: The reagent bottle used in this test should be dark colored to preserve the reagent from degradation.
  4. Dropper: It helps to transfer the reagent from the bottle to the test tubes while performing the Bial test.

Procedure of Bial’s Test

First tube: negative, second tube: negative and third tube: presence of pentose sugar. Source: Bial’s test

The steps for performing Bial’s test are as follows:

  1. Label the first test tube as a positive control, the second as a negative control, and the third as a test.
  2. Then, in the tube labeled as a positive control, add 1 ml xylose solution; in the negative control, add 1 ml distilled water; and in the tube labeled as the test, add 1 ml of the sample solution.
  3. After that, add 1 ml of Bial’s reagent in all the tubes.
  4. Place all the tubes in a water bath for 3-5 minutes.
  5. Finally, note the change in color in all the tubes.

Result Interpretation of Bial’s Test

The result from the Bial’s test are interpreted as follows:

Tested substances Observation Interpretation
Positive control (test tube with xylose) Color changes from light green to blue-green Positive Bial’s test
Negative control (test tube with water) No change in color Negative Bial’s test
Test (tube with samples) 1. Color changes to blue-green
2. Color changes to muddy brown		 1. Positive Bial’s test (Presence of pentose or pentose derivatives)
2. Negative Bial’s test (Absence of pentose or pentose derivatives)

Applications

The Bial’s test is used for analyzing carbohydrates in the samples. Following are the application of Bial’s test:

  1. The test helps differentiate pentose and pentose-derived carbohydrates from other carbohydrates.
  2. The Bial orcinol test, a modified version of this test, helps compute RNA concentrations.

Advantages

Like other tests for determining carbohydrates, this test has various advantages. Some of them are as follows:

  1. The test procedure is simple and easy to perform.
  2. This test is less time-consuming, i.e., analyzing a single analyte takes less than an hour.

Disadvantages

Like all procedures, this test also has some cons. Some of the shortcomings of this test are as follows:

  1. Prolonged heating can lead to false positive results due to the formation of gluconates. Hence care must be given to incubation time. Positive control also helps in mitigating this limitation.
  2. Different pentose sugars may give different colors; the intensity of the color does not correlate to the concentration of the sample.

References

  1. Mustansiriyah University. (2020). Bial`s Test. https://uomustansiriyah.edu.iq/media/lectures/6/6_2020_04_10!01_26_32_PM.pdf
  2. UNIVERSITY OF ANBAR. (n.d.). Bial’s test. https://www.uoanbar.edu.iq/eStoreImages/Bank/2063.pdf
  3. Tiwari, PhD, Anand. (2015). Practical Biochemistry: A Student Companion.