Enzymes -- Biology for Engineers

---

Enzymes: To convey that without catalysis life would not have existed on Earth

Introduction to Enzymes:

• Enzymes are protein molecules that act as catalysts in biochemical reactions.
• They are crucial for facilitating almost all cellular processes, ensuring they occur at rates necessary for life.

Role of Catalysis in Life:

• Catalysis: A process that increases the rate of a chemical reaction by lowering its activation energy. Enzymes achieve this through their unique structures and specific binding sites.
• Activation Energy: The energy required to initiate a chemical reaction. Enzymes lower this barrier, making reactions proceed faster.

==Evolutionary Perspective:==

• Origin of Life: The RNA world hypothesis posits that early life forms may have relied on RNA for both genetic information and catalysis. RNA molecules known as ribozymes could have been the first catalysts.
• Protein Enzymes: With the evolution of protein synthesis, enzymes became more efficient and diverse, supporting the complexity of life.

Importance of Enzymes in Biological Processes:

1. Metabolism:

   • Enzymes catalyze metabolic pathways, which are series of chemical reactions occurring within a cell.
   • Without enzymes, metabolic processes such as glycolysis, the Krebs cycle, and oxidative phosphorylation would occur too slowly to sustain life.

2. DNA Replication and Repair:

   • Enzymes like DNA polymerase and ligase are essential for DNA replication and repair.
   • These processes ensure the integrity of genetic information during cell division and in response to damage.

3. Protein Synthesis:

   • Enzymes such as RNA polymerase and ribosomal RNA play key roles in transcription and translation of DNA.
   • They ensure that proteins are synthesized accurately and efficiently, which is vital for cellular function and structure.

4. Cell Signaling and Regulation:

   • Enzymes like kinases and phosphatases modulate signaling pathways by adding or removing phosphate groups.
   • This regulation is crucial for cellular responses to external stimuli and maintaining homeostasis.

5. Digestion:

   • Digestive enzymes (e.g., amylase, protease, lipase) break down macromolecules in food into absorbable units.
   • This allows nutrients to be assimilated and utilized by the body.

---

Enzymology: How to monitor enzyme-catalyzed reactions

Monitoring enzyme-catalyzed reactions is crucial for understanding enzyme function, kinetics, and regulation. Various techniques are employed to observe and measure the progress of these reactions.

(No need to go way in depth)

1. Spectrophotometry

• Principle: Measures the change in absorbance or fluorescence of substrates or products.
• Types:
  • UV-Vis Spectrophotometry: Used when substrates or products absorb light at specific wavelengths. For example, NADH absorbs at 340 nm, allowing the monitoring of dehydrogenase reactions.
  • Fluorescence Spectroscopy: Some reactions produce fluorescent products. For example, monitoring the cleavage of a fluorogenic substrate by a protease.

2. Chromatography

• Principle: Separates reaction components based on their interactions with a stationary phase and a mobile phase.
• Types:
  • High-Performance Liquid Chromatography (HPLC): Separates and quantifies reaction components. For example, HPLC can separate glucose-6-phosphate and fructose-6-phosphate.
  • Gas Chromatography (GC): Used for volatile compounds, such as monitoring the conversion of fatty acids to esters.

3. Electrophoresis

• Principle: Separates macromolecules based on size and charge under an electric field.
• Types:
  • Polyacrylamide Gel Electrophoresis (PAGE): Used for proteins. For example, analyzing protein cleavage by a protease.
  • Agarose Gel Electrophoresis: Used for nucleic acids. For example, monitoring restriction enzyme activity on DNA.

4. Radioactive Labeling

• Principle: Incorporates radioactive isotopes into substrates to track reactions.
• Procedure: Uses isotopes like ^32P or ^14C. For example, ^32P-labeled ATP can be used to track kinase activity by detecting phosphorylated products.

5. Calorimetry

• Principle: Measures heat changes during reactions.
• Types:
  • Isothermal Titration Calorimetry (ITC): Measures heat released or absorbed during substrate binding. For example, studying enzyme binding and activity.
  • Differential Scanning Calorimetry (DSC): Analyzes thermal stability and enzyme-substrate interactions.

---

How does an enzyme catalyse reactions?

(Just focus on headings)

1. Active Site and Substrate Binding

Active Site:

• A specific region on the enzyme where the substrate binds.
• It is formed by the unique three-dimensional arrangement of amino acids.

Substrate Binding:

• The substrate binds to the active site, forming an enzyme-substrate (ES) complex.
• Binding involves non-covalent interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions.

2. Enzyme-Substrate Complex Formation

Specificity:

• Enzymes are highly specific to their substrates due to the precise fit between the active site and the substrate.
• Lock and Key Model: Substrate fits perfectly into the active site.
• Induced Fit Model: Enzyme changes shape to fit the substrate more snugly upon binding.

Transition State Stabilization:

• Enzymes stabilize the transition state of the reaction, lowering the activation energy needed for the reaction to proceed.

3. Mechanisms of Catalysis

Proximity and Orientation:

• Enzymes bring substrates close together and orient them correctly to facilitate the reaction.
• This reduces the entropy cost of aligning reactive groups.

Strain and Distortion:

• Enzymes can induce strain in the substrate, making it more reactive and easier to transform.

Microenvironment:

• The active site provides an optimal environment for the reaction, such as excluding water to prevent hydrolysis or creating an acidic or basic environment for proton transfer.

Catalytic Residues:

• Specific amino acids in the active site participate directly in the catalysis, acting as proton donors or acceptors or forming transient covalent bonds with the substrate.

Examples

Hexokinase:

• Function: Catalyzes the phosphorylation of glucose to glucose-6-phosphate.
• Mechanism: Binds glucose and ATP, induces a conformational change, stabilizes the transition state, and transfers a phosphate group from ATP to glucose.

Lysozyme:

• Function: Breaks down bacterial cell walls by hydrolyzing glycosidic bonds in peptidoglycans.
• Mechanism: Binds the substrate, distorts the glycosidic bond, stabilizes the transition state, and facilitates bond cleavage through catalytic residues.

---

Enzyme Classification

Enzymes are classified into different categories based on the types of reactions they catalyze. This classification helps in understanding their specific roles in biochemical pathways and their mechanisms of action.

1. Oxidoreductases

• Function: Catalyze oxidation-reduction (redox) reactions, where electrons are transferred from one molecule (the reductant) to another (the oxidant).
• Examples:
  • Dehydrogenases: Remove hydrogen atoms from a substrate (e.g., lactate dehydrogenase converts lactate to pyruvate).
  • Oxidases: Transfer electrons to oxygen (e.g., cytochrome c oxidase in the electron transport chain).

2. Transferases

• Function: Transfer functional groups (e.g., methyl, glycosyl, phosphoryl) from one molecule to another.
• Examples:
  • Kinases: Transfer phosphate groups from ATP to substrates (e.g., hexokinase phosphorylates glucose to glucose-6-phosphate).
  • Transaminases: Transfer amino groups from one amino acid to a keto acid (e.g., alanine transaminase transfers an amino group from alanine to alpha-ketoglutarate).

3. Hydrolases

• Function: Catalyze the hydrolysis of various bonds, such as ester, glycosidic, peptide, and phosphodiester bonds, by adding water.
• Examples:
  • Proteases: Hydrolyze peptide bonds in proteins (e.g., trypsin cleaves peptide bonds in proteins at the carboxyl side of lysine and arginine).
  • Lipases: Hydrolyze ester bonds in lipids (e.g., pancreatic lipase breaks down dietary fats into fatty acids and glycerol).

4. Lyases

• Function: Catalyze the addition or removal of groups to form double bonds or breaking of various chemical bonds by means other than hydrolysis and oxidation.
• Examples:
  • Decarboxylases: Remove carboxyl groups from substrates, producing carbon dioxide (e.g., pyruvate decarboxylase converts pyruvate to acetaldehyde and CO₂).
  • Aldolases: Split carbon-carbon bonds (e.g., aldolase in glycolysis splits fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate).

5. Isomerases

• Function: Catalyze the rearrangement of atoms within a molecule to form isomers.
• Examples:
  • Racemases: Convert one isomer into another (e.g., alanine racemase converts L-alanine to D-alanine).
  • Mutases: Transfer functional groups within a molecule (e.g., phosphoglucomutase converts glucose-1-phosphate to glucose-6-phosphate).

6. Ligases

• Function: Catalyze the joining of two molecules with the formation of a new chemical bond, usually coupled with the hydrolysis of ATP or another nucleoside triphosphate.
• Examples:
  • DNA Ligase: Joins DNA fragments together by forming phosphodiester bonds (e.g., DNA ligase seals nicks in the DNA backbone during replication and repair).
  • Synthetases: Form new bonds using ATP (e.g., glutamine synthetase catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia).

---

Why should we know enzyme kinetic parameters to understand biology?

1. Enzyme Regulation and Metabolism

Metabolic Pathways:

• Enzyme kinetics helps us understand how enzymes control the flow of metabolites through metabolic pathways.
• For example, the regulation of glycolysis and the citric acid cycle depends on the kinetic properties of key enzymes.

Regulatory Enzymes:

• Enzymes that regulate metabolic pathways often exhibit complex kinetics, such as allosteric regulation, where the binding of a molecule at one site affects the enzyme’s activity at another site.

2. Drug Design and Pharmacology

Enzyme Inhibition:

• Many drugs function as enzyme inhibitors. Understanding the kinetics of inhibition (competitive, non-competitive, uncompetitive) is crucial for drug design.

Pharmacokinetics:

• Enzyme kinetics informs how drugs are metabolized in the body. The rate at which enzymes metabolize drugs affects their efficacy and toxicity.

3. Genetic and Evolutionary Insights

Mutations:

• Mutations can affect enzyme kinetics, leading to diseases. Studying these changes helps in understanding disease mechanisms and developing treatments.
• For example, genetic mutations affecting the enzyme phenylalanine hydroxylase lead to phenylketonuria, a metabolic disorder.

Evolution:

• Enzyme kinetics can provide insights into the evolutionary adaptation of organisms. Enzymes evolve to optimize the kinetic parameters suited to an organism’s environment and metabolic needs.

4. Biotechnological Applications

Industrial Enzymes:

• Enzyme kinetics is crucial for optimizing enzymes used in industrial processes, such as in the production of biofuels, food processing, and biotechnology.

Enzyme Engineering:

• Understanding the kinetic parameters allows scientists to engineer enzymes with desired properties, such as increased stability or altered substrate specificity.