1. Biopolymers and Enzymes

what are the 9 functions of proteins?

• enzymatic catalysis - accelerate the rate of biochemical reactions

• transport - e.g. hemoglobin transports oxygen, transferrin Fe++, albumin etc.

• structural support - all cellular and extracellular structures contain proteins e.g. collagen and keratin

• signal transduction - receptors for cell communication

• regulation - of hormones, growth factors, cytokines

• defense - immune response via antibodies, lectins, blood clotting factors and complement

• muscle contraction - for motor function e.g. actin, myosin, tropomyosin, troponin

• energy formation - electron transport chain in mitochondria

• storage - ferritin stores iron, myoglobin stores oxygen

what is a heteroprotein?

• Protein variants of the same function but found in different species.

• Proteins that are composed of both polypeptides and a non-protein component called a prosthetic group

what is an isoprotein?

• Protein variants of the same function but different locations, different tissues or subcellular structure in the same individual

• example: muscle (MM) or heart (MB) creatine kinase (CK); mitochondrial and cytosolic alanine aminotransferase (ALT)

what is an alloprotein?

• Protein variants of different individuals of the same species, often as a result of variant alleles of a gene (polymorphisms)

what is a peptide?

• Short chains of amino acids, typically <50 AA

what is a polypeptide?

• 20-100 AA

• Longer chains of amino acids, which are essentially precursor molecules for proteins

• When they fold into functional three-dimensional structures, they become proteins

what is an oligopeptide?

• 2-20 AA

• e.g. glutathione 3AA, oxytocin 9AA

what is a protein?

• >100 AA

• Larger, functional biomolecules made up of one or more polypeptides

• When the polypeptide chain has folded into a specific structure and performs a biological function.

what is an amino acid?

Amino acids are the building blocks of proteins. Each amino acid contains:

• An amino group (-NH₂)

• A carboxyl group (-COOH)

• A hydrogen atom (H)

• An R group (side chain), which determines the amino acid’s properties.

The unique sequence and characteristics of amino acids dictate how a protein folds and functions

what is a proteogenic amino acid?

• those responsible for the formation of peptides and finally proteins

what is an essential amino acid and give examples of them?

• essential amino acids have to be taken in by nutrition e.g. Val, Leu, Ile, Lys, Met, Phe, Thr, Trp, His

• semi-essential amino acids are Arg, His

name the 5 aliphatic amino acids

Gly, Ala, Val, Leu, Ile

name the 2 hydroxy amino acids

Ser, Thr

name the 2 S-containing amino acids

Cys, Met

name the cyclic iminoacid

Pro

name the 2 aromatic amino acids

Phe, Tyr, Trp

name the 3 basic amino acids

Lys, Arg, His

+ve charged at pH 7

name the 2 acidic amino acids

Asp, Glu

-ve charged at pH 7

name the 2 amide-containing amino acids

Asn, Gln

name the polar uncharged hydrophilic amino acids

Ser, Thr, Cys, Tyr, Asn, Gln

name the nonpolar hydrophobic amino acids

Gly, Ala, Val, Leu, Ile, Met, Phe, Pro, Trp

what is protein conformation?

spatial arrangement of atoms in a protein (the folding and interactions)

what is the primary structure of proteins?

• The sequence of amino acids in a polypeptide chain

• linear formations

• weak hydrogen bonding

• weak hydrophobic forces between R groups

• weak electrostatic forces

• van der waals forces

describe the properties of the peptide bond

• Planarity: due to resonance between the nitrogen and carbonyl oxygen, prevents rotation around the bond

• Partial double-bond character: due to resonance, making it less flexible.

• Peptides only occur in cis- or trans conformation

• Trans configuration: minimizing steric hindrance between adjacent amino acid side chains

• Stability: resistant to hydrolysis under physiological conditions, though it can be broken by specific enzymes (proteases)

• Amide linkage between between carboxylic acid of one AA to the amine of the other AA

• Bonds on either site of the alpha-carbons can rotate which gives the final protein flexibility

what is the secondary structure of proteins?

• Local folding/conformation of the polypeptide chain into α-helices or β-sheets, stabilized by hydrogen bonds

• peptide bond

• α‑Helix:

  • Hydrogen bonds form between the C=O group of residue i and the N–H group of residue i+4.

  • This pattern creates a helical structure stabilized by these bonds along the length of the helix.

• β‑Sheet:

  • In β‑sheets, hydrogen bonds form between C=O groups and N–H groups on adjacent, often extended, strands.

  • These strands can be arranged in a parallel or antiparallel fashion, each with a slightly different hydrogen bonding pattern

• Van der Waals Interactions

• Electrostatic Interactions

describe the alpha helix

• A right-handed helix with 3.6 amino acids per turn

• Stabilized by hydrogen bonds between the carbonyl oxygen of one amino acid and the amide hydrogen of an amino acid four residues ahead in the chain

• R groups extend outward from the helix, allowing them to interact with the environment or other parts of the protein.

describe the beta sheet

• Composed of beta-strands, which are extended stretches of polypeptide chains that lie alongside one another

• Stabilized by hydrogen bonds between carbonyl oxygens and amide hydrogens of adjacent strands

• Can be parallel or antiparallel.

what is random coil?

• A polymer conformation in which the monomer subunits are oriented randomly while still being bonded to adjacent units

• A region of the protein that does not have a regular secondary structure (alpha-helix or beta-sheet)

• Flexible and less ordered, allowing for more movement or functional versatility

• It is not one specific shape but a statistical distribution of shapes for all the chains in the population of macromolecules

what are supersecondary structures of proteins?

• Supersecondary structures are relatively small, conserved arrangements of secondary structure elements that often occur as a unit in different proteins.

• They help define the folding pathway of proteins by providing a modular framework that is both stable and functional.

• These motifs can often be recognized in various proteins and are thought to facilitate the evolution of new protein folds by recombination of these building blocks.

• Folding Pathway: Supersecondary structures can form early during the folding process and act as nucleation points around which the rest of the protein structure can be organized.

• Functionality: Because these motifs are involved in forming the active sites or binding sites of proteins, their conservation is critical to the function of many enzymes and binding proteins.

• β‑Hairpin:
  Two antiparallel β‑strands connected by a tight turn, forming a simple and common motif in many proteins.

• Helix-Turn-Helix:
  Consists of two α‑helices separated by a short loop. This motif is particularly well-known in DNA-binding proteins, where one helix often interacts with the DNA.

• β‑Alpha‑β Motif:
  A β‑strand, followed by an α‑helix, and then another β‑strand. This arrangement is common in many enzyme active sites and helps in the formation of larger structures like β‑sheets or α/β barrels.

• Greek Key:
  A pattern typically seen in β‑sheet structures where four β‑strands fold over in a way that resembles a key pattern often seen in classical Greek art.

• Rossmann Fold:
  This motif typically consists of a series of alternating β‑strands and α‑helices and is often involved in binding nucleotides such as NAD(P)H.

what is a motif?

• Simple combinations of secondary structures that recur in many proteins

• These motifs often serve as functional units and are involved in binding or structural roles

• Describes the connectivity between secondary structural elements

• Examples include the helix-turn-helix, beta-alpha-beta loop, and Greek key motifs

what is a domain?

• sequence of the protein that can evolve, function and exist independendly of the rest of the protein chain forming a 3-D structure each

• Independently folding regions of a protein that have specific functions

• Domains are larger structural units than motifs and can combine multiple secondary structures

• Many proteins are modular, with distinct domains responsible for distinct functions, such as binding, catalysis, or regulation

what is the tertiary structure of proteins?

• The overall 3D shape of the polypeptide, formed by interactions between the R groups (side chains) of amino acids

• hydrophobic interactions

• peptide bond

• hydrogen bonds

• van der waals forces

• ionic bond/salt bridge

• disulfide bridges: covalent bonds that form between the sulfur atoms of cysteine side chains.

what is a fibrous protein?

• Polypeptide chains that are arranged in long strains or sheets that usually consist of a single type of secondary structure

• Have elongated, linear structures

• Serve structural roles by providing strength and stability

• Insoluble in water due to extensive hydrophobic regions

• Axial ratio more than 10

• Less sensitive to changes in pH and temperature

• Examples: Collagen (in connective tissue), keratin (in hair and nails), and elastin (in elastic fibers).

describe the structure of collagen

• An essential fibrous protein of connective tissue

• Each circuit is made up of 1050 AC

• The primary structure consists of 3 PPVs that contain AA

• sequences (Glycine-X-Y)n, where X and Y are any other AA

• but most commonly proline or OH-proline (hydroxy proline)

• less often lysine and OH-lysine.

what is a globular protein?

• Contain several types of secondary structures unlike its fibrous counterpart

• Compact, spherical structures

• Perform dynamic functions such as catalysis (enzymes), transport, regulation, immune protection etc

• Axial ratio less than 10

• More sensitive to changes in pH and temperature

• Soluble in water due to hydrophilic residues on the surface

• Examples: Hemoglobin (oxygen transport), myoglobin (oxygen storage), enzymes (like lysozyme), insulin and antibodies

• They contribute to many enzymes and proteins of the immune systems immunoglobulins!

describe the structure of myoglobin

• globular protein

• in the heart and skeletal muscles

• functions both as an oxygen reservoir and as an oxygen carrier, which increases the rate of oxygen transport in the cells of the muscle

• It consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of hemoglobin

what is the quaternary structure of proteins?

• Some proteins are made of multiple polypeptide chains (subunits).

• The arrangement of these subunits relative to each other forms the quaternary structure (e.g. hemoglobin is made up of four subunits)

describe the role of regulation for quaternary structure of proteins

• Allosteric Regulation:

  • Quaternary structures allow proteins to undergo allosteric changes, where the binding of a molecule (ligand) to one subunit can influence the activity of another subunit.

  • This is important for many enzymes and receptors.

  • For example, hemoglobin, which has four subunits, shows cooperative binding of oxygen, meaning when one subunit binds oxygen, the affinity of the remaining subunits increases.

• Protein-Protein Interactions:

  • Proteins with quaternary structures can interact with other proteins or molecules to form large complexes, such as in cell signaling pathways or immune responses.

  • The arrangement of subunits is key to the activation or inhibition of certain biological processes

• Functional Modulation:

  • Subunit composition can vary in some protein complexes, altering their functional capabilities.

  • Different arrangements or combinations of subunits can lead to varying protein functions, increasing the versatility and adaptability of the protein

what enzymes and chaperones are involved in formation of quaternary structure of proteins?

• Chaperone Proteins:

  • Molecular chaperones, like heat shock proteins (HSPs) and chaperonins, assist in the proper folding of nascent or denatured proteins.

  • They do not determine the final structure but help prevent incorrect interactions that could lead to misfolding.

  • Examples include:

    • Hsp70: Binds to newly synthesized or partially unfolded proteins, preventing aggregation.

    • GroEL/GroES (in bacteria): A chaperonin system that provides an isolated environment for folding.

• Protein Disulfide Isomerase (PDI):

  • Catalyzes the formation and rearrangement of disulfide bonds, which stabilize the protein structure, particularly in secretory proteins.

  • By catalyzing disulfide exchange, the rupture of an S—S bond and its reformation with a different partner cysteine, protein disulfide isomerase facilitates the formation of disulfide bonds that stabilize a protein's native conformation.

• Peptidyl-prolyl Isomerase (PPI): Helps the correct folding of proteins by catalyzing the cis-trans isomerization of proline residues, which can act as a rate-limiting step in folding.

• Proline – cis, trans - isomerases

  • All X-Pro peptide bonds, where X represents any residue, are synthesized in the trans configuration.

  • The cis configuration is particularly common in β-turns. Isomerization from trans to cis is catalyzed by the enzyme proline- cis, trans -isomerase

what are the mechanisms that maintain the conformation of proteins?

• Covalent Bonds: Disulfide bonds between cysteine residues help stabilize tertiary and quaternary structures

• Non-covalent Interactions: Hydrogen bonds, van der Waals forces, and hydrophobic interactions all play roles in maintaining protein conformation

• Chaperones and these enzymes ensure that proteins achieve and maintain their correct structure, essential for proper cellular function

medical importance of proteins - defects in receptors

• Diabetes insipidus is caused by a failed folding of a mutated V2 gene - the vasopressin receptor (ADH) gene

• Familial hypercholesterolemia is caused by a mutation in the low density lipoprotein receptor (LDL-R) gene

medical importance of proteins - diseases due to impaired conformation

• In Alzheimer`s disease, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets.

• Prion diseases (Creutzfeldt-Jakob disease in humans, and bovine spongiform encephalopathy (mad cow disease) in cattle; Prion diseases can be transmitted by the protein alone without involvement of DNA or RNA

medical importance of proteins - molecular diseases

• Sickle Cell Disease: A single amino acid change in hemoglobin (Glu to Val) causes hemoglobin to polymerize under low oxygen conditions, distorting the shape of red blood cells and impairing their function.

medical importance of proteins - defects in the post-translational modification of proteins

• Scurvy is a disease caused by a deficiency of vitamin C (ascorbic acid), which is essential for the hydroxylation of proline and lysine residues in collagen. This post-translational modification is vital for collagen stability and function. Without adequate vitamin C, collagen fibers become weak, leading to symptoms such as:

  • Gum disease: Weak connective tissues result in swollen gums and tooth loss.

  • Skin issues: Impaired collagen synthesis leads to skin lesions and bruising

• Glycated hemoglobin (HbA1c) is formed when glucose binds to hemoglobin in red blood cells. It serves as a critical biomarker for long-term glucose control in diabetes management. Elevated levels of HbA1c indicate poor glycemic control and are associated with complications such as:

  • Diabetic neuropathy: High HbA1c levels can predict nerve damage.

  • Cardiovascular diseases: Chronic hyperglycemia linked to elevated HbA1c increases the risk of heart disease

describe the structure of hemoglobin

• Tetrameric Structure: Hemoglobin is a heterotetramer composed of four polypeptide subunits:

  • Two Alpha Chains: These are derived from the α-globin gene family.

  • Two Beta Chains: These originate from the β-globin gene family.

  • This arrangement allows for cooperative binding of oxygen, enhancing its efficiency in oxygen transport

• Heme Group: Each subunit contains a heme group, which is an iron-containing compound that binds oxygen. The heme consists of:

  • Porphyrin Ring: A cyclic structure that coordinates the iron atom.

  • Iron Ion (Fe²⁺): This ion can reversibly bind to oxygen molecules, allowing hemoglobin to transport oxygen throughout the body

what is the isoelectric point (pl) of a protein?

• the pH at which the protein carries no net electrical charge

• at this point, the positive and negative charges on the protein balance out, and the protein becomes electrically neutral

• at physiological pH (pH=7.4) the protein molecules have different number of positively and negatively charged AA residues – the proteins appear as polyelectrolytes

what is protein precipitation?

• At the isoelectric point, proteins tend to be least soluble in aqueous solutions because they do not repel each other due to lack of net charge.

• As a result, protein precipitation can occur.

• Protein solubility depends on electrostatic repulsion.

• At the pI, since there's minimal repulsion, proteins aggregate and precipitate.

• This property is used in protein purification, where adjusting the pH to the protein's pI can cause it to precipitate from solution.

• At that pH – the molecule is electrically neutral and does not move (migrate) in electrophoresis

what is denaturation?

• the loss of a protein’s natural three-dimensional structure, leading to the disruption of its biological function

• can affect a protein’s secondary, tertiary, and quaternary structures, though the primary structure (sequence of amino acids) remains unchanged

what is electrophoresis?

• a technique used to separate charged molecules, like proteins or nucleic acids, based on their size and charge by applying an electric field across a gel or another medium

• allows separation and determination of proteins on the basis of their isoelectric points and molecular weight

• Electrophoresis of proteins is generally carried out in gels made up of cellulose acetate, agarose or of the cross-linked polymer polyacrylamide gel

• In electrophoresis, the force moving the macromolecule is the electrical potential

• The electrophoretic mobility of the molecule depends on the size and the shape of the molecules.

• Thus the migration of a protein in a gel during electrophoresis is a function of its size and its shape.

describe the electrophoresis of plasma proteins

• Serum protein electrophoresis (SPE) is a clinical technique used to separate and analyze different proteins in blood serum. The profile generated can provide important diagnostic information.

• Key Protein Fractions in Serum:

  • Albumin: The most abundant protein, important for maintaining osmotic pressure and transporting substances

  • Alpha (α) Globulins: Include proteins like α1-antitrypsin and haptoglobin, involved in anti-inflammatory and transport processes

  • Beta (β) Globulins: Include transferrin and complement proteins, involved in iron transport and immune function

  • Gamma (γ) Globulins: Mainly immunoglobulins (antibodies), which play a role in immune defense

describe the clinical applications of electrophoresis

• Multiple Myeloma: Characterized by a sharp spike in the gamma globulin region, known as an M-protein spike, due to overproduction of monoclonal immunoglobulins

• Liver Disease: Reduced albumin and increased gamma globulins are seen in chronic liver diseases

• Nephrotic Syndrome: There is often a significant reduction in albumin levels, with compensatory increases in alpha-2 globulin

• Acute Inflammation: Increases in α1 and α2 globulins are seen, as these are acute-phase proteins

what are the types of nucleic acids?

• DNA (Deoxyribonucleic Acid): Stores genetic information in cells and is passed on from generation to generation. DNA’s main role is to provide the instructions for synthesizing proteins and other cellular components.

• RNA (Ribonucleic Acid): Involved in the decoding of the genetic information stored in DNA and plays several roles in gene expression and protein synthesis. There are several types of RNA, including:

  • mRNA (messenger RNA): Carries the genetic message from DNA to the ribosome for protein synthesis

  • tRNA (transfer RNA): Delivers amino acids to the ribosome during protein synthesis

  • rRNA (ribosomal RNA): Part of the ribosome, which synthesizes proteins

what is the biological role of nucleic acids?

• Genetic Information Storage (DNA): DNA contains the instructions needed to create and regulate proteins and cellular activities

• Gene Expression (RNA): RNA translates the genetic code into proteins by interpreting the sequence of nucleotides in DNA

• Catalytic Activity (Ribozymes): Some RNA molecules (ribozymes) have enzymatic functions, facilitating reactions in cells, like RNA splicing.

describe the chemical composition of nucleic acids

Each nucleotide, the building block of nucleic acids, consists of three components:

• Nitrogenous Base: A purine (adenine, guanine) or a pyrimidine (cytosine, thymine in DNA, and uracil in RNA)

• Pentose Sugar: A five-carbon sugar – deoxyribose in DNA and ribose in RNA

• Phosphate Group: One or more phosphate groups attached to the sugar

describe the chemical bonds of nucleic acids

• hydrogen bonds between bases

• 3’-5’ phosphodiester bonds between nucleotides (sugar and phosphate group)

• glycosidic bond between base and sugar

describe the biological importance of free nucleotides

• play critical roles in cellular processes

• ATP (adenosine triphosphate) provides energy for various cell processes

• GTP (guanosine triphosphate) involved in protein synthesis and signal transduction

• cAMP (cyclic adenosine monophosphate) mediates the efforts of hormones

• NAD+ (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) is a coenzyme for many redox reactions

describe the features of polynucleotide chains

• Directionality: Polynucleotide chains have a distinct 5’ end (with a free phosphate group) and a 3’ end (with a free hydroxyl group). This 5’ to 3’ polarity is crucial for processes like DNA replication and transcription.

• Complementarity: In double-stranded DNA, the two polynucleotide chains are antiparallel and held together by complementary base pairing (A-T, G-C).

• Double Helix: In DNA, two polynucleotide chains coil around each other to form a double helix, with the sugar-phosphate backbone on the outside and the nitrogenous bases on the inside.

• Base Stacking: The nitrogenous bases stack above each other within the helix, stabilized by van der Waals forces and hydrophobic interactions

describe the Watson and Crick model of DNA

• described the double-helix structure of DNA, revolutionizing the understanding of genetic material

• Double Helix: DNA consists of two polynucleotide strands coiled around a central axis

• Antiparallel Strands: The two strands run in opposite directions (5’ to 3’ and 3’ to 5’)

• Base Pairing: Complementary bases (A-T and G-C) form hydrogen bonds, with A-T pairing by 2 hydrogen bonds and G-C by 3 hydrogen bonds, ensuring the stability of the structure.

• Right-Handed Helix: The DNA double helix is right-handed, with about 10 base pairs per turn.

• Major and Minor Grooves: The structure has a larger major groove and a smaller minor groove, important for protein-DNA interactions.

• This model explained how DNA could store genetic information and be accurately replicated.

what are the levels of organisation of DNA (by analogy with proteins)?

1. linear sequence of nucleotides

2. double helix

3. supercoiling of DNA

4. interactions with proteins e.g. histone proteins to form chromatin

Purine and Pyrimidine Analogues as Anticancer and Antiviral agents - General Mechanism of Action

• Incorporation into Nucleic Acids:

  • Mimicry and Misincorporation:
    These analogues can be taken up by cells and phosphorylated into their active nucleotide forms. Once activated, they may be incorporated into DNA or RNA during replication or transcription. Their incorporation can lead to faulty base pairing or chain termination.

  • Chain Termination:
    Some analogues lack the necessary chemical groups (such as a 3'-OH group) needed to form the next phosphodiester bond, resulting in premature termination of DNA or RNA synthesis.

• Enzyme Inhibition:

  • Targeting Polymerases and Kinases:
    Many of these analogues inhibit enzymes critical for nucleic acid synthesis. They can inhibit DNA or RNA polymerases directly or interfere with kinases responsible for activating the prodrug.

  • Disruption of Metabolic Pathways:
    By interfering with nucleotide biosynthesis, they can cause an imbalance in the nucleotide pool, further impairing DNA replication and repair.

• Induction of Apoptosis:

  • Cancer Cells:
    Rapidly dividing cancer cells are particularly vulnerable to disruptions in DNA synthesis. The damage induced by these analogues can trigger cell cycle arrest and apoptosis (programmed cell death).

• Selectivity and Toxicity:

  • Targeting Rapidly Dividing Cells:
    Both cancer cells and virus-infected cells often replicate their nucleic acids more rapidly than normal cells, making them more susceptible to these analogues. However, because normal proliferative cells (like those in the bone marrow, gastrointestinal tract, and hair follicles) can also be affected, side effects and toxicity are significant considerations.

• Activation and Resistance:

  • Prodrug Activation:
    Many analogues require activation by cellular or viral enzymes. Variability in enzyme expression can affect both efficacy and toxicity.

  • Resistance Mechanisms:
    Mutations in viral enzymes (such as thymidine kinase) or in cellular enzymes responsible for drug activation can lead to resistance, necessitating combination therapies or the development of new analogues.

• Therapeutic Windows:

  • The dosing and scheduling of these drugs are critical to maximize the killing of cancer or virus-infected cells while minimizing harm to normal cells.

Purine Analogues as Anticancer and Antiviral agents

Anticancer Applications:

• 6-Mercaptopurine (6-MP) and 6-Thioguanine:

  • Usage:
    These are used primarily in the treatment of leukemias, particularly acute lymphoblastic leukemia (ALL).

  • Mechanism:
    After metabolic activation, they become incorporated into DNA and RNA, leading to impaired nucleic acid function and triggering cell death.

• Fludarabine:

  • Usage:
    Employed in the treatment of chronic lymphocytic leukemia (CLL) and other hematological malignancies.

  • Mechanism:
    It interferes with DNA synthesis by inhibiting DNA polymerase and ribonucleotide reductase, resulting in apoptosis.

• Cladribine and Clofarabine:

  • Usage:
    Used in certain leukemias and lymphomas, these agents are particularly effective in disorders involving rapidly dividing lymphoid cells.

  • Mechanism:
    They are resistant to degradation by adenosine deaminase, accumulate in cells, and cause DNA strand breaks and apoptosis.

Antiviral Applications:

• Acyclovir (a guanosine analogue):

  • Usage:
    Widely used in the treatment of herpes simplex virus (HSV) and varicella-zoster virus infections.

  • Mechanism:
    Selectively phosphorylated by viral thymidine kinase, acyclovir’s active triphosphate form is incorporated into viral DNA, causing chain termination and inhibiting viral replication.

• Ganciclovir:

  • Usage:
    Primarily used against cytomegalovirus (CMV) infections, especially in immunocompromised patients.

  • Mechanism:
    Like acyclovir, it is phosphorylated by viral enzymes and then incorporated into viral DNA, leading to chain termination.

Pyrimidine Analogues as Anticancer agents

Anticancer Applications:

• 5-Fluorouracil (5-FU):

  • Usage:
    A mainstay in the treatment of various solid tumors, including colorectal, breast, and head and neck cancers.

  • Mechanism:
    5-FU is converted intracellularly into metabolites that inhibit thymidylate synthase, leading to a deficiency in thymidine required for DNA synthesis, as well as direct incorporation into RNA.

• Cytarabine (Ara-C):

  • Usage:
    Particularly important in the treatment of acute myeloid leukemia (AML) and other hematological malignancies.

  • Mechanism:
    It is incorporated into DNA during replication, where it inhibits DNA polymerase and results in chain termination.

• Gemcitabine:

  • Usage:
    Used in the treatment of pancreatic, lung, and breast cancers.

  • Mechanism:
    Once phosphorylated, gemcitabine is incorporated into DNA, causing masked chain termination and interfering with DNA repair mechanisms.

describe the primary structure of nucleic acids

• Each nucleotide, the basic building block of nucleic acids, is composed of three parts:

  • Phosphate Group:
    A phosphate group is attached to the 5′ carbon of the sugar and is responsible for linking adjacent nucleotides.

  • Pentose Sugar:

    • DNA: Contains deoxyribose.

    • RNA: Contains ribose.

  • Nitrogenous Base:
    There are different bases depending on the type of nucleic acid:

    • DNA: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).

    • RNA: Adenine (A), Uracil (U) instead of thymine, Guanine (G), and Cytosine (C).

• Phosphodiester Bonds:

  • Nucleotides are connected via phosphodiester bonds, which form when the phosphate group of one nucleotide reacts with the hydroxyl group on the 3′ carbon of the sugar in the next nucleotide.

  • This linkage gives the nucleic acid a directionality, typically denoted as 5′ (five prime) to 3′ (three prime).

  • The 5′ end has a free phosphate group, and the 3′ end has a free hydroxyl group.

describe the conformation of DNA

• B-DNA:

  • The most common form in cells.

  • It’s a right-handed double helix with about 10 base pairs per turn.

  • The major and minor grooves are distinct, and it is the biologically active form.

• A-DNA:

  • A more compact right-handed helix.

  • It is typically found in dehydrated conditions or in RNA-DNA hybrids

• Z-DNA:

  • A left-handed helix with a zigzag backbone, which may play a role in gene regulation and is typically found in regions rich in alternating purines and pyrimidines

describe the different types of RNA

• mRNA (Messenger RNA, 5% of RNA): single-stranded molecule that carries the genetic code from DNA to the ribosome

• tRNA (Transfer RNA, 15% of RNA): essential for decoding mRNA into amino acids during protein synthesis

• rRNA (Ribosomal RNA, 80% of RNA): forms complex secondary and tertiary structures, contributing to the ribosome's catalytic activity, provides the structural framework for ribosome assembly

• snRNA (Small Nuclear RNA): plays a role in splicing, forming part of the spliceosome, a complex that removes introns from pre-mRNA

• miRNA - micro RNA

• siRNA - small interfering RNA

what is a nucleosome and describe the structure of a nucleosome

• the basic unit of chromatin structure in eukaryotic cells, which helps in packaging DNA into the cell nucleus

• Nucleosomes consist of 147 base pairs of DNA wrapped around a core of eight histone proteins (two copies each of H2A, H2B, H3, and H4)

• Histone H1 binds the linker DNA between nucleosomes, further compacting the DNA

• Nucleosomes help condense DNA, reducing its overall length and allowing it to fit within the nucleus

• They regulate gene expression by controlling the accessibility of DNA to transcription factors and other regulatory proteins

describe histone proteins

• Histones (H2A, H2B, H3, and H4) play a crucial role in organizing DNA into nucleosomes

• They are positively charged, facilitating their interaction with the negatively charged DNA backbone

• Post-translational modifications (acetylation, methylation, phosphorylation) on histones can either tighten or loosen their interaction with DNA, thereby regulating gene expression

describe the non-histone proteins

• These include transcription factors, scaffold proteins, and DNA repair enzymes, which help maintain chromatin structure, regulate transcription, and facilitate processes like replication and repair

• They provide structural support and are involved in various chromosomal functions, including DNA replication, recombination, and segregation

describe the denaturation and renaturation of DNA

• Denaturation refers to the separation of double-stranded DNA into single strands due to the disruption of hydrogen bonds between base pairs.This can be caused by heat, extreme pH, or chemical agents (e.g., urea)

• Renaturation (also called reannealing) is the process where complementary DNA strands reassociate into a double helix when optimal conditions are restored

what is the hyperchromic effect?

• Upon denaturation, DNA absorbs more ultraviolet (UV) light, particularly at 260 nm, due to the unstacking of the bases.

• This increase in absorbance is known as the hyperchromic effect and is used to monitor DNA denaturation

describe the melting point of DNA

• The melting point of DNA is the temperature at which half of the DNA molecules in a solution are denatured (half are single-stranded, and half are double-stranded)

• GC-rich DNA has a higher melting point than AT-rich DNA because G-C base pairs have three hydrogen bonds, compared to two for A-T pairs, making them more stable

what are riboenzymes and their role in RNA maturation?

RNA molecules that have catalytic activity. They are involved in several important biological processes, including:

• RNA Splicing: Certain ribozymes, such as those found in the spliceosome, catalyze the removal of introns from pre-mRNA during RNA splicing

• Self-Splicing Introns: Some RNAs can catalyze their own splicing without proteins (group I and II introns)

• tRNA Processing: Ribozymes like RNase P cleave precursor tRNA molecules to form functional tRNA

describe post-transcriptional modification of mRNA

• 5 ' “Capping”:

  • The cap is a 7-methylguanosine attached to the 5 -terminal end of the mRNA through an unusual 5 '→5 ' triphosphate linkage that is resistant to most nucleases.

  • The addition of this cap helps stabilize the mRNA and permits efficient initiation of translation

• Addition of a poly-A tail:

  • Most eukaryotic mRNA have a chain of 40–250 adenine nucleotides attached to the 3 ' – end.

  • This poly-A tail is not transcribed from the DNA, but rather is added after transcription.

  • These tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation.

  • After the mRNA enters the cytosol, the poly-A tail is gradually shortened

• Removal of introns:

  • Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns) that do not code for protein.

  • The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA.

  • The process of removing introns and joining exons is called splicing.

  • The molecular complex that performs these tasks is known as the spliceosome

describe micro-RNAs and their role in the regulation of gene expression

• small RNA molecules that regulate gene expression in cells

• are able to recognize target messenger RNAs by sequence complementarity and regulate their protein translation

what are water-soluble vitamins?

• essential nutrients that dissolve in water and are not stored in the body

• B-complex vitamins and vitamin C

• crucial roles in various biological functions

• since they are water soluble (excess amounts are excreted from the body with water), we must get them daily

Vitamin B1 - active form, biological role, sources, avitaminosis

• Active form is thiamine pyrophosphate (TFF)

• Biological Role: essential for carbohydrate metabolism and nerve function

• Sources: nuts, seeds, whole grains

• Avitaminosis: Beriberi - muscle pain, heart failure

• Wernicke-Korsakov syndrome - often in alcoholics - acute manifestation of encephalopathy - general inadequate condition, ophthalmoplegia (paralysis of the muscles of the eye)

Vitamin B2 - active form, biological role, sources, enzyme, avitaminosis

• Active form: riboflavin

• Biological Role: Important for energy production and skin health. Precursor to coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in redox reactions

• Sources: eggs, fish, dairy, green leafy vegetables

• Avitaminosis: ariboflavinosis - sore throat, redness, swelling of the lining of the mouth and throat

Vitamin B3 - active form, biological role, sources, enzymes, avitaminosis

• Active form: nicotinamide and nicotinic acid

• Biological role: Aids in DNA repair and metabolism of fats and sugars. nicotiamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) cofactors of dehydrogenases and reductases respectively

• Sources: meat, fish, whole grains, rice

• Avitaminosis: pellagra - dermatitis, dementia, death if untreated

Vitamin B5 - active form, biological role, sources, avitaminosis

• Active form: pantothenic acid

• Biological role: synthesis of coenzyme A, fatty acid metabolism, Krebs cycle

• Sources: chicken, beef, oats, potatoes, tomatoes

• Avitaminosis: burning feet syndrome - fatigue

Vitamin B6 - active form, biological role, sources, enzyme, avitaminosis

• All forms: pyridoxine, pyridoxal, pyridoxal phosphate, pyridoxamine

• active form: pyridoxal phosphate PP

• biological role: amino acid metabolism and neurotransmitter synthesis. coenzyme of amino acid transaminases and decarboxylase

• sources: fish, chicken, eggs, non-citrus fruits

• avitaminosis: depression, seizures, hypochromic anemia, oxalate kidney stones, weakened immune system

Vitamin B7 - active form, biological role, sources, avitaminosis

• active form: biotin

• biological role: fatty acid synthesis and glucose metabolism

• sources: eggs, almonds, spinach, sweet potatoes

• avitaminosis: hair loss, skin rashes, neurological symptoms

Vitamin B9 - active form, biological role, sources, enzyme, avitaminosis

• active form: tetrahydrofolic acid THFA

• biological role: coenzyme for transferases, DNA synthesis and repair

• sources: leafy greens, seeds

• avitaminosis: megaloblastic anemia, neural tube defects in pregnancy

Vitamin B12 - active form, biological role, sources, avitaminosis

• active form: cobalamin

• biological role: transferases, red blood cell formation and neurological function

• sources: animal products, meat, dairy, eggs

• avitaminosis: pernicious anemia, megaloblastic anemia, depression, demyelination of nerves, increased Irisk of myocardial infarction and stroke

Vitamin C - active form, biological role, sources, avitaminosis

• active form: ascorbic acid

• biological role: collagen synthesis, antioxidant, immune function, wound healing, bone remodelling, cofactor of hydrolases

• sources: citrus fruits, strawberries, bell peppers, broccoli

• avitaminosis: scurvy - fatigue, swollen gums, joint pain, anemia

what are lipid-soluble vitamins?

• essential nutrients that dissolve in fats and oils

• vitamins A, D, E, and K (KADE)

• stored in the body's fatty tissues and liver, which allows for longer retention compared to water-soluble vitamins

Vitamin A - active form, biological role, sources, avitaminosis, hypervitaminosis

• active form: retinol

• biological role: vision, skin health, immune function

• sources: liver, fish oils, dairy products, colorful fruits and vegetables

• avitaminosis: night blindness, nyctalipia

• hypervitaminosis: nausea, headaches, blurred vision, dizziness, liver damage

Vitamin D - active form, biological role, sources, avitaminosis, hypervitaminosis

• active form: calciferol, D3 is 1,25-dihydroxy cholecalciferol

• biological role: regulates calcium and phosphorus metabolism, essential for bone health and immune function

• Along with the parathyroid hormone (parathormone, PTH) and calcitonin, vitamin D3 regulates the blood levels of calcium and phosphorus.

• sources: sunlight exposure, fatty fish, egg yolk

• avitaminosis: rickets in children (continued formation of the collagen matrix of bone, softening of bones) and osteomalacia (demineralization of preexisting bones increases their susceptibility to fracture) in adults (bone pain and muscle weakness)

• hypervitaminosis: hypercalcemia (high calcium levels), leading to nausea, weakness, kidney stones, and calcification of soft tissues

Vitamin E - active form, biological role, sources, avitaminosis, hypervitaminosis

• active form: α-tocopherol

• biological role: powerful natural antioxidant, protecting cells from oxidative damage; supports immune function

• sources: nuts, seeds, vegetable oils, green leafy vegetables

• avitaminosis: neurological problems, poor nerve conduction, muscle weakness and vision problems

• hypervitaminosis: may interfere with vitamin K's role in blood clotting, increasing the risk of bleeding

Vitamin K - active form, biological role, sources, avitaminosis, hypervitaminosis

• active form: phylloquinone

• biological form: antihemorrhagic, essential for blood clotting and bone metabolism

• sources: green leafy vegetables (kale, spinach), broccoli

• avitaminosis: increased bleeding tendencies due to

  impaired blood clotting; can lead to hemorrhagic disease in

  newborns

• hypervitaminosis: can interfere with anticoagulant medications and lead to clotting issues.

• Vitamin K undergoes reduction to hydroquinone with the participation of the enzyme Vitamin K epoxide reductase.

what are the characteristics of enzymes?

• biological catalysts that speed up biochemical reactions without being consumed or permanently altered in the process

• Highly Specific: Enzymes typically catalyze only one type of reaction or act on a specific substrate due to their unique active sites

• Efficient: They can accelerate reactions by factors of up to 10^6 to 10^12 compared to the uncatalyzed reactions

• Regulated: Enzymatic activity can be regulated through various mechanisms (e.g., allosteric regulation, covalent modification, or feedback inhibition)

• Mild Reaction Conditions: Enzymes function under physiological conditions, such as moderate temperatures, neutral pH, and aqueous environments, which differ from non-biological catalysts that often require extreme conditions

• Reusability: After catalyzing a reaction, enzymes are not consumed and can be reused repeatedly

what are ribozymes?

• A small fraction of RNA, called ribozymes, also act as biocatalysts.

• These are the RNAs that ensure splicing in mRNA maturation, and also 28S RNA has peptidyl transferase activity .

what are the similarities of enzymes to other catalysts?

• They increase the rate of only spontaneously occurring reactions by providing alternative route with lower activation energy

• They change the rate of the forward and reverse reactions without shifting the chemical equilibrium

• Act in insignificant amounts

• Remain chemically unchanged at the end of the reaction

what are the differences between the enzymes and the rest of the catalysts?

• Enzymes are highly effective - they speed up from 10^9 to 10^12 times

• The enzymes lower the activation energy because they carry out the reaction differently with lower energy requirements.

• It is characterized by the formation of an intermediate compound between the starting substance (so-called substrate) and the enzyme, which is called enzyme-substrate complex.

• Several intermediates may be formed.

• Regardless of how many intermediate phases there are, their activation energy is always lower than that of the uncatalyzed reaction

• The particular enzyme exhibits high specificity in terms of the nature of the reaction and the structure of the substrate

• They act in mild physiologic conditions- temperature less than 40°С, pH about 7, atmosphere pressure 1 atm

• The activity and amount are under precise regulation

describe the structure of enzymes

• simple - only protein

• complex/holoenzymes = protein and nonprotein part

• apoenzyme - protein part

• cofactor - non protein part - can be prosthetic groups or coenzymes

• prosthetic groups - small inorganic molecule tightly bound to apoenzyme

• coenzyme - large organic molecule loosely bound to apoenzyme

describe the 6 classes of enzymes

• Oxidoreductases: Catalyze redox reactions (e.g., dehydrogenases, oxidases)

• Transferases: Transfer functional groups from one molecule to another (e.g., kinases, aminotransferases, methyltransferases, and sulfotransferases).

• Hydrolases: Catalyze the hydrolysis of bonds (e.g., proteases, lipases, phosphatases and esterases).

• Lyases: Catalyze the addition or removal of groups to form double bonds (e.g., decarboxylases, aldolases, synthases and deaminases).

• Isomerases: Catalyze the rearrangement of atoms within a molecule (e.g., racemases, epimerases, isomerases and mutases).

• Ligases: Catalyze the joining of two molecules with the input of ATP (e.g., synthetases, carboxylases).

describe the mechanisms of enzyme catalysis

• E + S → ES → EP → E + P

• Proximity and Orientation: Enzymes bring substrates into close proximity and orient them correctly to increase the likelihood of a reaction

• Induced Fit: When a substrate binds to the active site, the enzyme undergoes a conformational change that tightens the binding and enhances the enzyme's catalytic activity. This is called the induced fit model, as opposed to the older lock-and-key model

• Transition State Stabilization: Enzymes stabilize the transition state of the reaction, making it easier for the reaction to proceed

• Covalent Catalysis: In some cases, the enzyme forms a temporary covalent bond with the substrate to facilitate the reaction

• Acid-Base Catalysis: Amino acid side chains in the active site can donate or accept protons (act as acids or bases) to stabilize reaction intermediates

• Metal Ion Catalysis: Some enzymes use metal ions (e.g., Zn²⁺, Mg²⁺, Fe²⁺) as cofactors to aid in catalysis by stabilizing charged intermediates or assisting in redox reactions.

what is activation energy?

• Activation energy is the amount of energy required to move all molecules of a reactant into an activated state

• The difference between the energy levels of the ground state and the transition state is the activation energy, ΔG

• For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state

• A higher activation energy corresponds to a slower reaction

describe the active site, amino acid residues in active sites and key features

• the region on an enzyme where the substrate binds and the reaction occurs ON surface of the enzyme not IN the enzyme

• It is typically a small part of the enzyme’s structure but plays a critical role in its function

• Catalytic – take part in the reaction

• Contact – take part in binding the substrate to AS

• Assistant (additional) – assist the catalytic and contact groups

• Conformational – associated in folding of the 3D structure of enzymes

• Specificity:

  • The active site has a precise arrangement of amino acids that determine which substrates can bind.

  • The shape and chemical environment of the active site are complementary to the substrate.

  • Enzymes whose substrates or products are optically active substances exhibit stereospecificity

• Binding: The substrate is held in the active site through non-covalent interactions like hydrogen bonding, hydrophobic interactions, van der Waals forces, and ionic bonds

• Microenvironment: The active site can provide a unique microenvironment, such as a hydrophobic region or specific pH, that facilitates the reaction

describe the different types of enzyme specificity

• Absolute Specificity: The enzyme acts on a single substrate. For example, urease catalyzes only the hydrolysis of urea

• Group Specificity: The enzyme acts on substrates with a particular functional group, such as alcohol dehydrogenase, which works on alcohols

• Linkage Specificity: The enzyme targets a specific type of bond, such as proteases, which break peptide bonds

• Stereochemical Specificity: The enzyme distinguishes between different stereoisomers (e.g., D- and L- forms of amino acids or sugars). Lactate dehydrogenase acts only on the L-isomer of lactate.

what is enzyme kinetics?

• the rates at which enzymatic reactions occur and how those rates are affected by factors like substrate concentration, enzyme concentration, temperature, and pH

• the central aspect of enzyme kinetics is the relationship between an enzyme, its substrate, and the formation of products