Mar 31

Copyright Information

• This lecture presentation and accompanying PowerPoint slides are exclusively copyrighted by Professor Omri.

• Usage is limited to students enrolled in Biochemistry I (CHMI-2227 E) in the Winter term 2026 at Laurentian University.

• Unauthorized or commercial use of the lectures, including uploading to external sites, is strictly prohibited.

Enzyme Inhibition

• Enzyme inhibition refers to the reduction or elimination of catalytic activity of an enzyme.

Useful Drugs as Enzyme Inhibitors

• Several drugs function as enzyme inhibitors:
    - Lovastatin:
      - Used to treat hypercholesterolemia.
      - Blocks the synthesis of cholesterol.
      - Inhibits HMG-CoA reductase (3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A reductase).
    - Aspirin:
      - Prevents arachidonic acid from being converted into prostaglandins and thromboxanes.
      - Acts through inhibition of the enzyme cyclooxygenase.
    - 5-Fluorouracil (5-FU):
      - Inhibits the enzyme thymidylate synthetase, which converts dUMP to dTMP.
      - Provides thymine necessary for DNA synthesis.
      - Employed as a chemotherapeutic agent to inhibit cancer cell proliferation.

Types of Enzyme Inhibition

• Enzyme inhibitors can be classified into:
    - Reversible Inhibitors:
      - Bind to an enzyme to inhibit its activity, but can be removed.
      - Types include:
        - Competitive Inhibitor:
          - Blocks access to the active (catalytic) site by mimicking the substrate (substrate analogue).
        - Noncompetitive Inhibitor:
          - Binds to a site different from the active site, causing a change in the conformation of the enzyme, thus inhibiting activity.
    - Irreversible Inhibitors:
      - Inhibition cannot be reversed, often involving the formation or breaking of covalent bonds on or to the enzyme.

Competitive Inhibition Effects

• Competitive inhibitors do not affect Vmax but increase Km (Michaelis constant).

• Represented graphically on a double-reciprocal plot as follows:
    - Intersection point on vertical axis corresponds to $rac{1}{V_{max}}$.

Noncompetitive Inhibition Effects

• Noncompetitive inhibitors do not alter Km, but decrease Vmax.

• Graphical representation on a double-reciprocal plot:
    - Intersection point on horizontal axis corresponds to $- rac{1}{K_m}$.

Visualization of Inhibition Types

• Competitive Inhibition:
      - Plots of $rac{1}{V}$ vs. $rac{1}{[S]}$ at various inhibitor concentrations intersect at the same point on the vertical axis ($1/V_{max}$).

• Noncompetitive Inhibition:
      - Plots of $rac{1}{V}$ vs. $rac{1}{[S]}$ at varying inhibitor concentrations intersect at the same point on the horizontal axis ($- rac{1}{K_m}$).

Allosteric Enzymes

• Allosteric: Derived from Greek, where "allo" means other and "steric" refers to shape.

• Definition: Allosteric enzyme is an oligomer whose biological activity is influenced by the binding of substrates to sites other than the active site.
    - These substances alter the enzyme’s activity by changing the conformations of its quaternary structure.

Allosteric Effectors

• Allosteric Effector: A substance that modifies the behavior of an allosteric enzyme, which can be an:
    - Allosteric Inhibitor
    - Allosteric Activator

Introduction to the Chemistry of Nucleic Acids

• 1868: Friedrich Miescher isolated a molecule called Nuclein from salmon sperm and pus of wounds.
    - Nuclein contains phosphorus and is soluble in water, precipitating in a light acidic medium.
    - Extracted using ether and acidic digestion (with trypsin) for protein precipitation.
    - This represented the first description of DNA.

Milestones in DNA Research

• 1910: Dr. Hoppe-Seyler's students discovered the components of DNA (bases and sugar).

• 1924: Robert Feulgen discovered a method for specific coloration of DNA using fuchsine dye.

• 1930: Levene analyzed DNA components:
    - Identified four nitrogen bases: Cytosine, Thymine, Adenine, Guanine.
    - Recognized the components sugar (deoxyribose) and phosphate group.

• 1928: Griffith discovered the phenomenon of bacterial transformation.

• 1944: Avery, MacLeod, and McCarty demonstrated that DNA acts as the transforming agent.

• 1947: Chargaff reported that in a DNA molecule the quantities of A equals T and G equals C.

• 1952: Hershey and Chase showed DNA injection by T2 bacteriophage.

• 1953: Watson and Crick proposed the double helix model of DNA, suggesting it as the genetic information carrier.

• 1958: Meselson and Stahl proved DNA replication via a semi-conservative method.

• 1965: Nirenburg, Leder, and collaborators identified the genetic code for protein synthesis from DNA.

• 1979: Alexander Rich's MIT team discovered Z-DNA, a left-handed, zigzagging DNA structure.

Griffith's Experiment on Bacterial Transformation (1928)

• Griffith conducted an experiment using Pneumococcus bacteria and mice.

• Established initial evidence that specific chemicals within cells serve as genetic material.

Experimental Design

• Strains of Pneumococcus:
    1. S strain (smooth, virulent, polysaccharide-coated).
    2. R strain (rough, non-virulent, not polysaccharide-coated).

Results of the Experiment

• Injected live S strain into mice, resulting in sickness and death.

• Injected heat-killed S strain, observing that mice remained healthy.

• Injected live R strain, and mice showed no signs of infection.

• Following the injection of a mixture of heat-killed S strain and live R strain, the mice died.
    - Isolated live S strain from the blood of deceased mice.

Conclusions Drawn by Griffith

• Concluded that the live R strain bacteria absorbed genetic material from the dead S strain.

• Suggested that the transforming substance in heat-killed bacteria was likely the gene for virulence.

• Identified the missing piece of the puzzle to be the chemical nature of the transforming substance.

Identifying the Transforming Material

• In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty provided clarity on the transforming substance using a transformation test similar to Griffith's.

• Process included removal of proteins from the heat-killed S-strain extracts, confirming transformation still occurred.

• Enzymatic treatments (trypsin, chemotrypsin, RNAse) showed no effect on transformation ability.

• DNAse treatment destroyed the transforming capacity of the virulent extract, leading to the conclusion that the transforming material was DNA.

Chemical Structure and Base Composition of DNA

• 1952: Erwin Chargaff analyzed base ratios in DNA across various organisms.
    - Observed equal quantities of A and T, G and C in DNA, forming Chargaff’s Rule.
    - Total amount of purines (A + G) equaled total amount of pyrimidines (C + T).

Methods for Analyzing DNA Composition

• Investigated DNA base composition via hydrolysis using formic acid and observed base separation through paper chromatography.

Classification of Nucleic Acids

• Nucleic acids are categorized into two main types:
    1. Deoxyribonucleic Acid (DNA): Contains information for amino acid sequences, organized into genes.
    2. Ribonucleic Acid (RNA): Functions in the cellular mechanisms linking amino acids in a specific sequence.

Nucleotide Structure

• Nucleic acids are biopolymers made up of nucleotides composed of:
    - Sugar (monosaccharides D-ribose or 2-deoxy-D-ribose).
    - Organic Base (heterocyclic aromatic amine from purine or pyrimidine).
    - Phosphate.

Nucleosides

• A nucleoside contains:
    1. Sugar (D-ribose or 2-deoxy-D-ribose).
    2. Organic base (either purine or pyrimidine).
    - Lacks phosphate group.

Nucleotide Bonds

• Nucleotides connect in polynucleotides through phosphodiester bonds, forming:
    - Sugar-Phosphate Backbone:
      - -C-O-R
      - -O\text{-P-O-R}
      - -O\text{H}

Structural Characteristics of Polynucleotides

• A polynucleotide consists of bases along the sugar-phosphate backbone with specific ends:
    - 5' End: Terminating at the phosphate group.
    - 3' End: Terminating at the hydroxyl group.

Sugar Types in Nucleic Acids

• Pentose Sugar:
    - RNA: b-D-Ribofuranose.
    - DNA: b-D-2-Deoxyribofuranose.

Nitrogenous Bases in DNA and RNA

• For DNA:
    - Purines: Adenine (A), Guanine (G).
    - Pyrimidines: Thymine (T), Cytosine (C).

• For RNA:
    - Purines: Adenine (A), Guanine (G).
    - Pyrimidines: Uracil (U), Cytosine (C).

Numbering Conventions

• Purines (adenine, guanine) are numbered counterclockwise; pyrimidines (cytosine, thymine, uracil) are numbered clockwise.

Properties of Bases

• Both purines and pyrimidines are weak bases, capable of forming proton equilibria.

• Their planar structure allows absorption of UV light at 260 nm.

Tautomerism in Bases

• Tautomerism involves proton exchange between atoms in a molecule, commonly between keto and enol forms.

• Keto-enol tautomerism is a specific form of tautomerism related to nitrogenous bases.

Nucleosides and their Nomenclature

• Nucleosides featuring ribose are called ribonucleosides.

• Those with deoxyribose are termed deoxyribonucleosides.

• Atoms in sugars and bases are labeled with primed and regular numbering conventions respectively.

Nucleotide Formation

• A nucleotide forms when phosphoric acid esterifies to a hydroxyl group on a nucleoside.

• Ester formation can occur at the:
    - 2’, 3’, or 5’ positions for ribonucleosides.
    - 3’ or 5’ positions for deoxyribonucleosides.

Nomenclature of Nucleotides

• Nomenclature is based on the nucleoside name plus the position and number of phosphate groups:
    - Examples:
      - Deoxyadenosine 5'-monophosphate (dAMP).
      - Deoxyadenosine 5'-diphosphate (dADP).
      - Deoxyadenosine 5'-triphosphate (dATP).

• Phosphates are denoted as alpha (α), beta (β), and gamma (γ) based on their position.

Functions of Nucleotides

• Nucleoside 5'-triphosphates serve as energy carriers.

• Bases function as recognition units in biological processes.

• Cyclic nucleotides act as signal molecules to regulate cellular metabolism and reproduction.

• Specific roles include:
    - ATP is central to energy metabolism.
    - GTP drives protein synthesis.
    - CTP is involved in lipid synthesis.
    - UTP facilitates carbohydrate metabolism.