Enzymes and Proteins - Grade 11 Biology

Enzymes

Unit Learning Outcomes

• Upon completion of this unit, students will be able to:
  • Explain enzymes: their properties, factors affecting their activities, functions, mechanisms of action, regulation, industrial applications, and kinetics.
  • Demonstrate proteins and their structures.

What are Enzymes?

• At the end of this section, students will be able to:
  • Define enzymes and activation energy.
  • Explain how enzymes work.
  • Describe the catalysis reaction of enzymes, activities, and substrates.

Definition

• Enzymes are protein molecules acting as biological catalysts (biocatalysts).
• They accelerate the rate of chemical reactions by lowering activation energy.
• Activation energy is the minimum energy required for reactants to be converted into products.
• All cells contain different enzymes depending on the type of living cell, engaging in biochemical activity called metabolism.
• Metabolic processes in cells require enzymes to catalyze biochemical reaction types at rates fast enough to sustain life.

Enzyme Action

• Enzymes act upon molecules (substrates), converting them into products, and remain unchanged.

Energy

• Requires Energy (ATP) / Endergonic
• Generates Energy (ATP) / Exergonic

Properties and Functions of Enzymes

• After successfully completing this section, students will be able to:
  • Identify the properties of enzymes.
  • Explain the action of each property.
  • Describe the functions of properties.

General Properties

• The general properties of enzymes include their physical and chemical nature.
• They neither affect the nature of products formed nor undergo any changes by the reaction catalyzed.

A. Physical Properties of Enzymes

• The physical properties of enzymes include:
  1. Denaturation
  2. Solubility
  3. Colloids
  4. Biocatalysts
  5. Precipitation
  6. Molecular weight
  7. Enzyme activity

1) Denaturation

• Denaturation is the process of breaking the intra- and inter-molecular non-covalent bonds that distort the shape and active site of the enzymes.
• Enzymes are denatured by high heat (above 40ºC), alteration in pH (too low or too high), heavy metals and high salt concentrations, solvents, and other reagents.

2) Solubility

• Solubility is the property of enzymes that allows them to be dissolved in water, diluted glycerol, and alcohol, causing denaturation.

3) Colloidal Nature

• The colloidal nature of an enzyme is its tendency to have little or no dialysis across a semipermeable membrane due to its large size or high molecular weight.

4) Biocatalyst Property

• The biocatalyst property is the activity of enzymes in which very small quantities or a small amount of enzyme is enough to convert a large quantity of substrate and remain unchanged after the reaction.

5) Enzyme Precipitation

• Enzyme precipitation is the separation of enzymes for analysis using different aqueous or ethanol solvents.

6) Molecular Weights

• Molecular weights of enzymes are large protein biomolecules that hold polypeptide chains of various amino acid sequences, resulting in enzymes having a high molecular weight.

7) Enzymatic Activity

• Enzymatic activity is the general catalytic property of an enzyme, dependent on factors such as temperature, pH, enzyme concentration, and substrate concentration.
• Enzymes show the highest activity at optimum temperature and pH; a low concentration of enzymes and substrates slows down the enzymatic reaction.

B. Chemical Properties of Enzymes

• Enzyme chemical properties are:
  1. Sensitivity
  2. Regulations
  3. Specificity
  4. Catalysis
  5. Reversibility reactions

1) Heat and pH Sensitivity

• Heat and pH sensitivity is an enzymatic reaction to heat (temperatures) and pH (acidity and basicity), activated at optimum levels.

2) Regulation

• Regulation is the process of controlling the activity of enzymes by activator and inhibitor molecules.
• Enzyme regulation refers to the multiple mechanisms available to control the activity of enzymes.

3) Catalysis

• Catalysis is the process of accelerating a chemical reaction by a catalyst.
• Enzymes can transform about 100-10,000 substrates per second.
• The reactions catalyzed by enzymes show a $10^3$ - $10^8$ times faster reaction rate compared to non-catalyzed reactions.

4) Reversibility

• Reversibility is the ability of enzymatic biomolecules to catalyze various metabolic (anabolic and catabolic) reactions.
• It is the reaction to synthesize (build up new molecules or products) and decompose (break down different products) in which enzymatic reactions catalyze biochemical reactions in both forward and reverse directions.

5) Enzyme Specificity

• Enzyme specificity is a property of the enzyme that describes how restrictive the enzyme is in its choice of substrate.
• A completely specific enzyme would have only one substrate.

The Function of Enzymes

• Enzymes help speed up chemical reactions in the human body.
• They are essential for respiration, digesting food, and liver, muscle, and nerve function.
• Each cell in the human body contains thousands of enzymes that help facilitate chemical reactions within each cell.
• The turnover number of molecules is the number of substrates converted by one enzyme molecule per second at saturated (fully occupied) active sites.

Enzymes as Markers of Disease States

• Enzymes are markers of various diseases (blood tests; the rise or fall of enzyme levels can aid in the diagnosis of a variety of conditions) like:
  • Myocardial infraction: happens when part of the heart muscle doesn't get enough blood.
  • Jaundice: a sign of a problem with the liver, gallbladder, or pancreas.
  • Pancreatitis: inflammation of the pancreas may happen when digestive juices or enzymes attack the pancreas.
  • Cancer
  • Neurodegenerative disorders: many of these diseases are genetic; sometimes the cause is a medical condition such as alcoholism, a tumor, or a stroke.

Enzyme Active Sites

• Each enzyme has an active site with a unique shape that speeds up metabolism or chemical reactions in our bodies and builds substances in all living things.
• Enzyme acts on a substrate to produce product releasing enzyme for further use through steps 1-4.
• Enzymes perform their function by lowering a reaction's activation energy.
• Activation energy is the energy required to start a reaction.
• The lower the activation energy, the faster a reaction happens, e.g., enzymatic reactions between glucose and oxygen.

Some Enzymes in the Body and Their Functions

Protein Structures

Learning outcomes

• After the successful completion of this section, the student will be able to:
  • Explain the structure of proteins.
  • Determine the protein's primary, secondary, tertiary, and Quaternary structure
  • List the levels of protein function

Amino Acids and Peptides

• Proteins are very large molecules – macro-biopolymers – made from monomers called amino acids.
• Amino acids can be joined via a peptide bond
• An amino acid consists of an α-carbon atom bound to four groups:
  1. Amino group, $—NH<em>2$, $—$ exist as $NH</em>3^+$ under physiologic conditions).
  2. Carboxylic acid group, $—COOH$ (exist as $—COO^−$ under physiologic conditions).
  3. Simple hydrogen atom
  4. Commonly denoted "—R" and is different for each amino acid.

Protein structure.

• In the above sections, we have discussed that enzymes are proteins.
• Proteins have different structures.
• Protein structure is a polymer of amino acids joined by peptide bonds with three-dimensional arrangements of atoms in amino acid chain molecules.
• The protein complex macromolecules have four structural levels:
  1. Primary structure
  2. Secondary structure
  3. Tertiary structure
  4. Quaternary structure

1) The primary structure of proteins

• It is the sequence of amino acids linked together to form a polypeptide chain through peptide bonds created during the protein biosynthesis process
• Proteins with fewer than 50 sequences are peptides, and proteins with longer than 50 sequences of amino acids are polypeptides.

2) The secondary structure of proteins

• The secondary structure of a protein is a folded structure formed within a polypeptide due to interactions between atoms of the backbone based on hydrogen bonding and containing a-helix and ß-sheet types of strands

2.1 The α – Helix

• The α-helix is a right-handed coiled strand
• The α–Helix structure is one of the most common ways in which a polypeptide chain forms all possible hydrogen bonds by twisting into a right-handed screw with the NH group of each amino acid residue hydrogen-bonded to the CO of the adjacent turn of the helix.

2.2 ß–pleated sheet

• The hydrogen bonding in the ß-sheet is between the inter-strands and intra-strands in which the sheet conformation of the ß-sheet consists of pairs of strands lying side by-side.
• All peptide chains stretch out to nearly maximum extension, laid side by side and held together by intermolecular hydrogen bonds forming pleated folds of drapery

3. The tertiary structure of proteins

• The tertiary protein structure is the three dimensional shape of protein molecules that bend and twist to achieve the maximum stability or the lowest energy state.
• It is fashioned by many stabilizing forces due to the bonding interactions between the side-chain groups of amino acids

4. The quaternary structure of proteins

• A protein quaternary structure is the arrangement of multiple folded protein subunits in a multi-subunit complex.
• It is the association of several protein chains or subunits into closely packed arrangements with their own primary, secondary, or tertiary structures and held together by the hydrogen bonds Hemoglobin showing the quaternary structure

Enzyme substrate models

Learning outcomes

• After the successful completion of this section, the student will be able to:
  • Identify enzyme substrate models
  • Explain the active site of enzymes

Models for enzyme substrate interaction

• Enzyme substrate models are models for enzyme substrate interaction describing that the shapes of the active site and the substrate complement to fit into the binding active site perfectly.
• There are two different enzyme-substrate binding models
  1. lock and key model
  2. induced fit model

1. lock and key model

• The lock and key model is when enzyme active sites fill-in with a substrate to interact through non-covalent interactions.
• The model explains how the enzymes must bind to substrates before they catalyze a chemical reaction.
• Once the reaction progresses to the transition state and forms products, the active site will not be able to accommodate changes

2. Induced fit model

• when the active site of an enzyme is not perfect to perform the required function.
• The amino acid side chains that make up the active site mold into the precise positions enable the enzyme to perform its catalytic functions.
• The concept of induced fit states that when a substrate binds to an enzyme, it brings about a change in the shape of the enzyme, which either enhances or suppresses the activity of the enzyme.

Enzyme regulation

Learning outcomes

• After the successful completion of this section, the student will be able to:
  • Explain enzyme regulation
  • Distinguish activator and inhibitor enzymes
  • Describe substrate and bonding

Enzyme regulation as a control system

• Enzyme regulation is a control system for enzymatic activities in which enzymes are turned “on” or “off” depending on the organisms need.
• It adjusts enzymatic activities by other molecules to either increase or decrease the activities.
• It requires an extra activation process to pass through some modifications and functions.

Types of regulatory enzymes

• Regulatory enzymes are of two types,
  • Allosteric enzymes
  • Covalently modulated enzymes

1. Allosteric enzymes

• enzymes that have additional binding sites for effector molecules other than the active site that cause conformational changes, leading to changes of catalytic properties.

• Allosteric enzymes contain two binding sites called

  • active site/catalytic site for binding substrates
  • allosteric site/regulatory site for binding effectors

• Effectors are small molecules (inhibitor or activator) change the enzyme activity and function through reversible non-covalent binding of a regulatory metabolite in the allosteric site or non-active site.

• Effectors lead to conformational changes in a actual part of the enzyme that affect the overall conformation of the active site, causing modifications in the activity of the reaction.

2. Genetic and covalent modification

• The genetic and covalent modification modifies the protein surface and facilitates intracellular delivery.

• Genetic modification of enzymes is to improve the properties of enzymes and gain active and inactive forms.

• Covalent modulated enzymes are active and inactive forms of the enzymes altered due to covalent modification of structures catalyzed by other enzymes.

• Covalent modifications are enzyme-catalyzed alterations of synthesized proteins by the addition or removal of chemical groups.

• Modifications can target a single type of amino acid or multiple amino acids and will change the chemical properties of the site.

• Enzyme regulation occurs by the addition or elimination of some molecules attaching to the enzyme protein.

Enzyme inhibition

• Enzyme inhibition is a decrease in enzyme activity by enzyme inhibitors.
• Enzyme inhibitors are molecule that binds to an enzyme and blocks its activity.
• There are two types; these are
  • Reversible inhibitors
  • Irreversible inhibitors

Types of inhibitors.

• Reversible inhibitor: inactivates an enzyme through noncovalent easily reversed interactions. It is characterized by a rapid dissociation of the enzyme–inhibitor complex.
• Irreversible inhibitor: is a substance that permanently blocks the action of an enzyme. Usually covalently modify an enzyme, and inhibition can therefore not be reversed

Types of reversible inhibitors.

• Reversible inhibitors can be
  1. Competitive inhibitor is a molecule that blocks the binding of the substrate to the active site.
  2. Noncompetitive inhibitor binds to the enzyme already bound the substrate and decreases the effectiveness of the enzyme.
  3. Uncompetitive inhibitor binds only to the enzyme – substrate complex, but not to the free enzyme. The uncompetitive inhibitor’s binding site is created only when the enzyme binds the substrate.

Types of enzymes

Learning outcomes

After the successful completion of this section, student will be able to:

• List types enzymes
• Describe the function of each type of enzyme
• Define activities of each type of enzyme

Enzyme structural classification

• The structural classification of enzymes deals with the separation of an enzyme into
  1. simple proteins (active), contain only a polypeptide chain of linked amino acids
  2. conjugated proteins (holoenzymes), contain non-amino acid components
• Then, the conjugated protein (holoenzyme) is divided into two parts
  • The protein part (apoenzyme: inactive) responsible for the specificity of enzymes to their substrates.
  • The non-protein part (cofactor) necessary for the catalytic function of the enzymes. E.g. - Inorganic molecules metal ions ($Mg^{2+}$, $Fe^{3+}$, $Zn^{2+}$),
    • organic molecules or coenzymes ($NAD^+$, $NADP^+$, $FAD^{2+}$)
• Finally, the non-protein part (cofactor) separates into
  • Firmly attached metal ion (prosthetic group)
  • Loosely attached mostly vitamines (coenzyme) groups

Basic classification of enzymes

• Enzymes are composed of six classes based on

  • what and how they react,
  • The types of reactions they catalyzed,
  • Most enzymes are named for their substrates and for the reactions that they catalyze, with the suffix “ase” added.
    • Peptide hydrolase is an enzyme that hydrolyzes peptide bonds
    • ATP synthase is an enzyme that synthesizes ATP.

• The followings are basic classes of enzymes.

  1. Oxidoreductases are a class of enzyme that catalyzes oxidation-reduction reactions.

     • It catalyzes the transfer of electrons from one molecule (oxidant) to other molecule (reductant) reactions in the following pattern:

     • where A is the oxidant and B is the reductant.

  2. Transferase is an enzyme that transfers functional groups (like methyl) from one donor molecule to acceptor molecule.

  3. Hydrolases are enzymes that catalyze the hydrolysis of various bonds by reaction with/addition of water

  4. Lyases are enzymes that cleave bonds by other means rather than hydrolysis or oxidation in which two or more substrates are involved in one reaction (an addition or elimination reaction).

     • What is the difference between lyase and hydrolase?
     • Hydrolases are able to break chemical bonds, while lyases create new bonds by removing or adding functional groups and usually forms a new double bonds

  5. Isomerases are a general class of enzymes that catalyze reactions involving a structural rearrangement of a molecule in which bonds are broken and formed

  6. Ligases (synthetases) are enzymes that catalyze the joining of two molecules with concomitant hydrolysis of the di-phosphate bond in ATP.

Factors affecting enzyme action

Learning outcomes

After the successful completion of this section, student will be able to:

• List factors affecting enzyme actions
• Describe how each factor affects enzyme action
• Discuss on how to optimize the factors

Factors affecting enzyme action

• Enzymes work best within specific temperature and pH ranges and at optimal conditions (the condition under which particular enzyme is most active)
• An increase or decrease in the conditions of these factors affects the functions of enzymes.
• There are varieties of factors that affect the activity of enzymes:

Description on factors affecting enzymatic actions

1. Temperature: while all enzymes work best within the specific ranges of optimum temperatures, low or high temperature causes an enzyme to lose its activity and ability to bind a substrate and denatured. (Denaturation means a protein loses its shape)

   • Once enzymes denatured, they cannot be renatured.

   • Optimal temperature. As you increase temperature, the kinetic energy of reactants increases. This increases the rate in two ways:

     • More frequent collisions – the reactant particles move faster, collide more often with enzymes
     • More successful collisions -leading to a reaction

   • As you increase or decrease temperature further,

     • bonds in the active site begin to break
     • the tertiary structure is disrupted & this changes the specific shape of the active site
     • so it may no longer be complementary to the substrate.

2. pH: enzymes function at optimum pH (the potential of hydrogen ions) that ranges from too low (strong acid) to too high (too alkaline) pH.

   • Such extreme pH cause an enzyme to lose its ability to bind into a substrate.

   • Optimal pH. Most proteins and enzymes optimally function at a natural pH of around 7.

   • However, certain enzymes can tolerate higher or lower pH levels.

   • For example, the human protein, pepsin, which is found in the stomach, works best at a pH of 2, which is highly acidic.

   • Denaturation can occur at low or high pH.

   • The enzyme is affected due to disruption of the ionic and hydrogen bonds in the tertiary structure, which leads to an alteration in the specific shape of the active site.

3. Substrate concentrations: enzymes require a maximum limit of substrate concentration to bind.

   • Increasing concentration of substrate initially increases rate of reaction Since it increase the rate of collisions, so there will be more successful collisions per second. (assuming a constant enzyme concentration)
   • After a while the enzyme active sites are saturated.
   • After a certain point (the saturation point), increasing the concentration of a substrate, while keeping the enzyme concentration constant, no longer increases the rate of reaction.

4. Enzyme Concentration

   • Increasing [enzyme] initially increases rate.
   • increasing the number of enzymes increases the rate by increasing the amount of collisions between enzymes and substrates.
   • After a certain point, if the amount of substrate is kept constant, the rate of the reaction will not increase with increasing enzyme concentration.
   • If the supply of substrate is unlimited, addition of enzymes will continue to result in increased reaction rates. (The dotted line represents a reaction with unlimited substrate)

5. Radiation damages enzyme activities by reducing in enzymatic efficiency and creating disorders in the macromolecules.

6. Water: affects the performance of enzymes’ activity beyond its optimum level.

7. The salt concentration: Each enzyme has an optimal salt concentration. Changes in the salt concentration may also denature enzymes.

8. End product (Feedback) inhibition is a cellular control mechanism in that the end product inhibit enzyme's activity.

   • In feedback inhibition, the endproduct binds to the allosteric site of the enzyme and change the structure of the active site and this prevents the enzyme to perform its activity.