Doctors Revision

Doctors Revision Uganda

Enzymes: Enzymology & Kinetics

What are Enzymes?

Enzymes are biological catalysts that are predominantly protein in nature. They are specialized macromolecules that accelerate the rate of biochemical reactions within living organisms without being consumed in the process.

Precisely:

  • Biological: This highlights that enzymes originate from and function within living systems (cells, organisms).
  • Catalysts: A catalyst is any substance that increases the rate of a chemical reaction without undergoing any net change itself. Enzymes achieve this by providing an alternative reaction pathway with a lower activation energy.
  • Predominantly Protein in Nature: The majority of known enzymes are proteins. It's important to note the "predominantly" because there are exceptions, such as ribozymes (RNA molecules with catalytic activity), but for the purpose of general understanding, enzymes are equated with proteins.
  • Accelerate the Rate of Biochemical Reactions: Enzymes can speed up reactions by factors of millions or even trillions. Without enzymes, most biological reactions would occur too slowly to sustain life. For example, the hydrolysis of urea by the enzyme urease occurs 10¹⁴ times faster! (100,000,000,000,000)
  • Without Being Consumed in the Process: A defining characteristic of any catalyst is that it is regenerated at the end of the reaction. This means a single enzyme molecule can catalyze the transformation of many substrate molecules.

Are biological catalysts, proteins in nature, made in the body of living things whose function is to catalyze chemical reactions in living cells. So that reactions occur at a rate compatible with cellular processes.

Enzymes operate under specific conditions such as pH, temp, [S] etc..

Explain Enzyme Function

  • How do they speed up reactions?
  • How does it affect the energy of activation of a reaction?
  • Energy of activation: Energy needed for molecules to react with one another
  • Catalyst: Substance that increases the rate of a chemical reaction.

From the graph, we have REACTANTS and we have PRODUCTS.

Activation energy is the energy required to change Reactants into Products.

Usually, it takes A LOT of energy to change Reactants into Products, BUT THIS TIME ROUND, Enzymes act as CATALYSTS, and Catalysts are substances that lower the energy of activation needed for a reaction to occur.

Enzymes as Biological Catalysts: Lowering Activation Energy

The primary function of enzymes is to accelerate the rate of biochemical reactions by lowering the activation energy (Ea) of the reaction.

To understand this, let's first consider the concept of activation energy:

  • Activation Energy (Ea): For any chemical reaction to occur, reactant molecules must overcome an energy barrier. This barrier is the activation energy – the minimum amount of energy required to convert reactants into products. Think of it like pushing a ball over a hill.
  • Transition State: At the peak of this energy barrier, the reactants are in an unstable, high-energy intermediate state called the transition state. They are neither fully reactants nor fully products.

How Enzymes Lower Activation Energy:

Enzymes do not change the overall thermodynamics of a reaction (i.e., they do not change the equilibrium constant or the net energy change, ΔG, between reactants and products). Instead, they provide an alternative, lower-energy pathway for the reaction to proceed.

How?

  • Bringing Reactants Together (Proximity and Orientation): Enzymes have a specific region called the active site, which is a three-dimensional cleft or pocket where the reactant molecule(s), known as the substrate(s), bind. By binding to the active site, the enzyme brings the substrates into close proximity and holds them in the correct orientation to react.
  • Straining Substrate Bonds (Induced Fit): When the substrate binds, the enzyme often undergoes a slight conformational change, a phenomenon known as induced fit. This induced fit can subtly distort or strain specific bonds within the substrate, pushing it towards the unstable transition state.
  • Providing an Optimal Microenvironment: The active site can create a favorable microenvironment. This might involve:
    • Optimal pH: Certain amino acid side chains can act as acid or base catalysts.
    • Excluding Water: In some cases, excluding water can prevent unwanted side reactions.
  • Temperature: Increasing the temperature makes molecules move faster but biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway, “a shortcut”, which occurs with less energy requirement.

Analogy: Think of climbing over a mountain (high activation energy). An enzyme doesn't change the height of the valleys (reactants and products), but it digs a tunnel through the mountain (provides a lower activation energy pathway), making it much easier and faster to get to the other side.

  • Substrate: molecule that an enzyme acts upon to catalyze a chemical reaction.

Enzyme Structure

  • Enzymes are proteins.
  • They have a globular shape.
  • Have a complex 3-D structure.

Enzymes are globular proteins with specific three-dimensional shapes that are made to function as biological catalysts. This structure includes a specialized region called the active site, which is where the enzyme binds to its specific substrate molecule to catalyze a reaction.

The Protein Nature of Enzymes (Primary, Secondary, Tertiary, Quaternary Structure)

  • Primary Structure: This is the linear sequence of amino acids linked by peptide bonds, determined by the gene encoding the enzyme. It dictates how the protein will fold.
  • Secondary Structure: Localized, regular folding patterns of the polypeptide chain. The most common are:
    • Alpha-helices (α-helices): Spiral structures.
    • Beta-sheets (β-sheets): Extended, pleated structures.
  • Tertiary Structure: The three-dimensional shape of a single polypeptide chain. This intricate shape is stabilized by various interactions: Hydrogen & Ionic bonds, Disulfide bridges, and Hydrophobic interactions. This unique tertiary structure creates the specific active site and is essential for the enzyme's function.
  • Quaternary Structure: This applies to enzymes composed of more than one polypeptide chain (subunits). Not all enzymes have a quaternary structure.

The integrity of the 3D structure is essential for enzyme activity. Changes to this structure (e.g., denaturation) will lead to a loss of function.

Simple Enzymes vs. Conjugated Enzymes

Enzymes can be categorized based on their composition:

  • Simple Enzymes: These enzymes are composed entirely of protein. Example: Urease, pepsin, trypsin.
  • Conjugated Enzymes (Holoenzymes): Many enzymes require a non-protein component for their activity.
    • A conjugated enzyme in its active form is called a Holoenzyme.
    • The protein part is called the Apoenzyme.
    • The non-protein part is called a Cofactor.

Cofactors, Coenzymes, and Prosthetic Groups

The non-protein components:

  • Cofactor: This is a general term for any non-protein chemical compound required for the enzyme's activity. Inorganic Cofactors include metal ions like Mg²⁺ (for hexokinase), Zn²⁺ (for carbonic anhydrase), and Fe²⁺ or Fe³⁺ (for cytochromes).
  • Coenzyme: This is a type of cofactor that is a complex organic molecule, often derived from vitamins. Coenzymes act as carriers of specific functional groups, atoms, or electrons.
    • Examples:
      • NAD⁺ (from Niacin, B3) and FAD (from Riboflavin, B2) carry electrons.
      • Coenzyme A (CoA) (from Pantothenic Acid, B5) carries acyl groups.
      • Pyridoxal Phosphate (PLP) (from Vitamin B6) carries amino groups.
      • Biotin (from Vitamin B7) carries CO₂.
      • Tetrahydrofolate (THF) (from Folate, B9) carries one-carbon units.
  • Prosthetic Group: This is a type of cofactor that is tightly and stably (often covalently) bound to the apoenzyme. It does not dissociate during catalysis.
    • Examples:
      • Heme: A porphyrin ring with an iron atom, found in enzymes like catalase and cytochromes.
      • FMN (Flavin Mononucleotide): Can be a prosthetic group in some flavoproteins.

Summary of Enzyme Components:

Component Description
Apoenzyme The protein part of a conjugated enzyme (inactive on its own)
Cofactor General term for a non-protein chemical compound required for enzyme activity
— Coenzyme Organic cofactor, often loosely bound, acts as a carrier (derived from vitamins)
— Prosthetic Group Cofactor (organic or inorganic), tightly/covalently bound to the apoenzyme
Holoenzyme The complete, catalytically active enzyme (Apoenzyme + Cofactor)

Enzyme Specificity

Enzyme specificity refers to the ability of an enzyme to bind only certain substrates and catalyze only certain reactions. This characteristic is fundamental to the highly ordered and regulated nature of metabolism. Without specificity, enzymes would indiscriminately catalyze multiple reactions, leading to cellular chaos.

Enzymes exhibit various degrees of specificity, ranging from absolute (acting on only one molecule) to broad (acting on a class of molecules). This specificity arises from the unique three-dimensional structure of the enzyme's active site, which is complementary to its specific substrate(s).

Types of Enzyme Specificity:

  • Absolute Specificity: This is the highest degree of specificity. The enzyme acts on only one specific substrate molecule and catalyzes only one specific reaction.
    Example: Urease catalyzes the hydrolysis of urea but will not act on other related compounds like methylurea. Another example is succinate dehydrogenase, which only acts on succinate.
  • Group Specificity: The enzyme acts on a group of structurally related substrates that possess a specific functional group or chemical bond.
    Example: Hexokinase phosphorylates various 6-carbon sugars (e.g., glucose, fructose). Trypsin cleaves peptide bonds where the carbonyl group is donated by Lysine or Arginine residues.
  • Linkage (Bond) Specificity: The enzyme acts on a particular type of chemical bond, regardless of the structure of the rest of the molecule.
    Example: Lipases hydrolyze ester bonds in fats. Amylase hydrolyzes α-1,4-glycosidic bonds in starch, but not β-1,4-glycosidic bonds in cellulose.
  • Stereochemical (Optical) Specificity: The enzyme distinguishes between stereoisomers (molecules that differ in the 3D orientations of their atoms). Enzymes act on only one of two enantiomers (mirror image forms).
    Example: L-amino acid oxidase will only act on L-amino acids, not D-amino acids. Similarly, enzymes in carbohydrate metabolism can distinguish between D-glucose and L-glucose.

Models of Enzyme-Substrate Interaction (Explaining Specificity):

Two principal models describe how an enzyme's active site interacts with its substrate to achieve this specificity:

The "Lock and Key" Model (Emil Fischer, 1894):

  • Concept: This model proposes that the enzyme's active site has a rigid, pre-formed shape that is perfectly complementary to the shape of the substrate, much like a specific key (substrate) fits into a specific lock (enzyme).
  • Implication: Only the correct substrate can bind because its shape precisely matches the enzyme's.
  • Strengths: Explains the high degree of specificity observed in many enzymes.
  • Limitations: This model is somewhat static and doesn't fully account for the dynamic nature of enzymes or how they facilitate the transition state.

The "Induced Fit" Model (Daniel Koshland Jr., 1958):

  • Concept: This more refined model suggests that the active site is not rigid but rather flexible. When the substrate binds, it induces a conformational change in the enzyme. This change causes the active site to reshape itself, forming a tighter and more precise fit around the substrate, like a glove molding to a hand.
  • Implication: This induced change often brings the catalytic groups of the enzyme into optimal alignment to perform the reaction and is crucial for stabilizing the transition state.
  • Strengths:
    • Better explains how enzymes can facilitate the formation of the transition state.
    • Accounts for group specificity and for allosteric regulation.
  • Limitations: More complex to visualize than the lock and key model.

Relationship between the Models:

The "induced fit" model is largely accepted as a more accurate representation, with the "lock and key" model being a useful simplification. The initial binding might be somewhat "lock and key" like, but the subsequent conformational changes are "induced fit."

In essence, the precise three-dimensional architecture of the active site, sculpted by the enzyme's protein structure, is the molecular basis for its remarkable specificity. This ensures that cells can carry out a vast array of chemical reactions in a highly controlled and efficient manner.

Mechanism of Enzyme Action

Enzymes are catalysts that lower activation energy, right?
And that they do so with remarkable specificity. Right?

Now, let's see the step-by-step process of an enzyme-catalyzed reaction and see the ways they achieve this reduction in activation energy.

Steps in an Enzyme-Catalyzed Reaction:

    1. Enzyme-Substrate Binding (E + S ⇌ ES): The reaction begins when the substrate (S) molecule(s) bind to the specific region on the enzyme called the active site.
    • This binding is non-covalent (e.g., hydrogen bonds, ionic interactions) and is highly specific, as described by the "induced fit" model.
    • The formation of the Enzyme-Substrate complex (ES) is the first step.
     2. Formation of the Transition State (ES ⇌ ES‡): Once bound, the enzyme's catalytic power comes from its ability to stabilize the transition state (ES‡) – a high-energy, unstable intermediate where bonds are being broken and formed.
    • The enzyme facilitates this by orienting the substrates correctly, straining substrate bonds, creating a favorable microenvironment, and directly participating in the reaction.
     3. Enzyme-Product Formation and Release (ES‡ → EP → E + P): As the reaction progresses, the substrate is transformed into product(s) (P), forming an Enzyme-Product complex (EP).
    • The product(s) have a weaker affinity for the active site than the substrate.
    • Once formed, the product(s) are released.
     4. Enzyme Regeneration (E + P → E): The enzyme (E) is regenerated unchanged at the end of the reaction. It is now free to bind another substrate molecule.
    • This regeneration is what allows a single enzyme molecule to catalyze many thousands or millions of reactions per second.

How Enzymes Lower Activation Energy:

The active site of an enzyme is a highly sophisticated molecular machine that employs several strategies to lower the activation energy (Ea):

  • Proximity and Orientation Effects: By binding substrates in close proximity and holding them in the optimal orientation, enzymes dramatically increase the effective concentration of reactants, making the reaction far more likely to occur.
    Analogy: Imagine trying to find a specific key in a dark, messy room vs. having it handed to you in the correct orientation.
  • Bond Strain and Distortion (Induced Fit): The enzyme can induce a conformational change that stretches or bends specific bonds within the substrate, weakening them and making them more susceptible to breaking. This distortion resembles the transition state.
    Analogy: Bending a stick slightly before snapping it – the initial bend makes it easier to break.
  • Acid-Base Catalysis: Amino acid side chains within the active site (e.g., histidine, aspartate) can act as proton donors (acids) or acceptors (bases), stabilizing charged intermediates that form during the reaction.
    • General Acid Catalysis: Donating a proton to the substrate.
    • General Base Catalysis: Abstracting a proton from the substrate.
  • Covalent Catalysis: A nucleophilic amino acid residue in the active site can temporarily form a covalent bond with the substrate, creating a transient covalent enzyme-substrate intermediate. This redirects the reaction pathway to a lower-energy route.
    Analogy: A "relay race" where the enzyme temporarily "holds" part of the molecule.
  • Metal Ion Catalysis: Metal ions (cofactors like Zn²⁺, Mg²⁺) in the active site can participate by orienting the substrate, stabilizing charged transition states, or mediating redox reactions by gaining or losing electrons.

All these strategies converge to effectively reduce the energy barrier (activation energy) that reactants must overcome. By providing an energetically favorable pathway, enzymes dramatically increase reaction rates, enabling the chemistry of life to proceed at a functional pace.

Factors Affecting Enzyme Activity

The ability of an enzyme to catalyze a reaction is highly sensitive to its environment. Changes in certain physical and chemical factors can impact an enzyme's structure and, subsequently, its function.

1. Temperature:

  • Effect: Temperature has a dual effect on enzyme activity.
    • Low Temperatures: As temperature increases from low levels, the rate of enzyme-catalyzed reactions generally increases. This is because increased kinetic energy leads to more frequent collisions between enzyme and substrate.
    • Optimal Temperature: Each enzyme has an optimal temperature at which it exhibits maximum activity. For most human enzymes, this is around 37°C (body temperature).
    • High Temperatures: Beyond the optimal temperature, enzyme activity rapidly decreases. High temperatures cause denaturation, the irreversible loss of the enzyme's specific three-dimensional structure. The active site is destroyed, and the enzyme can no longer function.
  • Graphical Representation: A plot of enzyme activity versus temperature shows a bell-shaped curve, rising to a peak at the optimum and then sharply falling.

2. pH (Hydrogen Ion Concentration):

  • Effect: pH significantly affects the ionization state of amino acid residues in the enzyme, particularly those in the active site.
  • Optimal pH: Each enzyme has an optimal pH at which its activity is maximal. This pH corresponds to the state where the active site's amino acid residues have the correct charge to bind the substrate and facilitate catalysis.
  • Deviations from Optimal pH: Both very high and very low pH values can lead to denaturation. Changes in pH alter the charges on amino acid side chains, disrupting the ionic and hydrogen bonds that maintain the enzyme's 3D structure.
  • Graphical Representation: Enzyme activity versus pH produces a bell-shaped curve. The optimal pH varies depending on the enzyme's physiological location (e.g., pepsin in the stomach has an optimum pH of ~1.5-2.5, while trypsin in the small intestine has an optimum pH of ~7.5-8.5).

3. Substrate Concentration ([S]):

  • Effect: Assuming a fixed enzyme concentration, increasing the substrate concentration generally increases the reaction rate up to a certain point.
    • Low [S]: At low substrate concentrations, the reaction rate is directly proportional to the substrate concentration (first-order kinetics).
    • High [S] (Saturation): Eventually, all active sites become saturated with substrate. At this point, the enzyme is working at its maximum capacity, and adding more substrate will not increase the rate. The reaction reaches a maximum velocity (Vmax), and the kinetics become zero-order with respect to the substrate.
  • Graphical Representation: A plot of reaction rate versus substrate concentration shows a hyperbolic curve, rising steeply at first and then leveling off as Vmax is approached.

4. Enzyme Concentration ([E]):

  • Effect: Assuming a sufficient and non-limiting substrate concentration, the rate of an enzyme-catalyzed reaction is directly proportional to the enzyme concentration.
  • If you double the amount of enzyme, you double the number of available active sites, and thus, you double the maximum rate at which the reaction can proceed.
  • Graphical Representation: A plot of reaction rate versus enzyme concentration shows a linear relationship.

5. Presence of Activators and Inhibitors:

  • Activators:
    • Effect: Activators are substances that increase enzyme activity. They can do this by binding to the enzyme to enhance substrate binding, changing the enzyme's conformation to a more active form, or acting as essential cofactors (e.g., metal ions).
    • Example: Chloride ions (Cl⁻) are activators for salivary amylase.
  • Inhibitors:
    • Effect: Inhibitors are substances that decrease or stop enzyme activity. They are crucial for regulating metabolic pathways and are the basis for many drugs and poisons.
    • Inhibitors can block the active site, alter the enzyme's conformation, or bind irreversibly to the enzyme.
    • Examples: Heavy metals (lead, mercury) are often irreversible inhibitors. Many therapeutic drugs are enzyme inhibitors (e.g., penicillin inhibits bacterial cell wall synthesis enzymes).

Understanding these factors allows us to predict and control enzyme behavior. Maintaining optimal conditions is good for enzyme function in biological systems, and manipulating these factors is key in industrial applications and medical treatments.

Enzyme Kinetics

Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes. It involves measuring the rates of these reactions and investigating the factors that affect them. The goal is to understand the molecular mechanisms by which enzymes operate, and how their activity is regulated.

1. Understanding Vmax (Maximum Reaction Velocity) and Km (Michaelis Constant)

These two parameters are central to enzyme kinetics, particularly within the framework of the Michaelis-Menten model.

  • Vmax (Maximum Reaction Velocity): Vmax represents the maximum rate at which an enzyme-catalyzed reaction can proceed when the enzyme is fully saturated with substrate. At Vmax, all active sites of all enzyme molecules are occupied by substrate, and the enzyme is working at its absolute peak capacity.
    • Significance: It reflects the turnover number (kcat) of the enzyme, which is the number of substrate molecules converted to product per enzyme molecule per unit time, when the enzyme is saturated.
    • Unit: Expressed as concentration per unit time (e.g., micromoles per minute).
    • Vmax, Maximum Velocity: How fast the enzyme can work when it is completely flooded with substrate. Like the enzyme's top speed.
  • Km (Michaelis Constant): Km is the substrate concentration at which the reaction rate is half of Vmax (i.e., V = Vmax / 2).
    • Significance - Affinity: Km is often interpreted as an inverse measure of the enzyme's affinity for its substrate. A low Km indicates a high affinity (the enzyme binds tightly). Conversely, a high Km suggests a low affinity (the enzyme doesn't bind as tightly).
    • Physiological Relevance: In many biological systems, the substrate concentration is often near the Km value, allowing the enzyme's activity to be very responsive to changes in substrate concentration.
    • Km or Michaelis Constant: The substrate concentration needed to make the enzyme work at half of its speed.
    • Unit: Expressed as a concentration (e.g., micromolar, millimolar).

REMEMBER, “Low Km, Loves the Substrate”.
A low Km means the enzyme needs only a little bit of substrate to go fast, so it has a high affinity.
A high Km means it needs a lot of substrate, so it has a low affinity.

2. The Michaelis-Menten Equation

The relationship between the initial reaction rate (V₀), substrate concentration ([S]), Vmax, and Km is described by the Michaelis-Menten Equation:

V₀ = (Vmax * [S]) / (Km + [S])

Where:

  • V₀ = initial reaction velocity (rate)
  • Vmax = maximum reaction velocity
  • [S] = substrate concentration
  • Km = Michaelis constant

...but how did we get to this equation?

Michaelis-Menten Equation (Step-by-Step)

This is a guide to deriving the equation. Each step includes a real-world analogy to make the concepts easier to grasp.

Step 1: The Main Goal & The Key Players

Enzyme kinetics is the study of how fast enzymes work. Our goal is to create a formula that predicts the speed (or velocity, V) of the reaction based on how much "stuff" (or substrate, [S]) we give the enzyme.

The Key Players:

  • E = Enzyme: The worker or machine.
  • [S] = Substrate: The raw material.
  • [ES] = Enzyme-Substrate Complex: When the enzyme is holding the substrate, ready to work.
  • P = Product: The finished item.
E + S ⇌ ES → E + P

Analogy: The Pizza Shop

  • Enzyme (E) is the Pizza Chef.
  • Substrate ([S]) is the Dough.
  • Enzyme-Substrate Complex ([ES]) is the Chef holding the Dough.
  • Product (P) is the finished Pizza.

The faster the chef can make pizzas (V), the more customers we can serve! Our goal is to find out how the amount of dough we have ([S]) affects the speed of pizza making.

Step 2: Reaction Speed Depends on the "Busy-ness" of the Enzyme

The speed of our reaction (V) is directly related to how many enzymes are currently busy. In other words, the speed is proportional to the concentration of the Enzyme-Substrate Complex ([ES]).

V = k_p * [ES]

Here, k_p (also called k_cat) is a constant representing how fast one enzyme can convert the substrate into a product once it's holding it.

Analogy: Pizza Making Speed

The rate of pizza production (V) depends on how many chefs are actively making pizzas ([ES]). k_p is like the personal speed of each chef (e.g., each chef can make 2 pizzas per minute).

Step 3: Total Enzyme vs. Free Enzyme

At any time, the total number of enzymes ([E]total) is split into two groups: those that are free ([E]) and those that are busy with a substrate ([ES]).

[E]total = [E] + [ES]

We can rearrange this to find the amount of free enzyme:

[E] = [E]total - [ES]

Step 4: Vmax - The Absolute Maximum Speed

If we have a huge amount of substrate, all our enzymes will be busy all the time. The reaction can't go any faster. This top speed is called Vmax. Since all enzymes are busy, [ES] is equal to [E]total.

Vmax = k_p * [E]total

Analogy: Maximum Pizza Output (Vmax)

If there is an infinite supply of dough, all our chefs ([E]total) will be making pizzas nonstop. The rate of production at this point is the maximum possible, Vmax.

Step 5: The Dissociation Constant (Ks) - How "Clingy" is the Enzyme?

The dissociation constant, Ks, tells us about the binding relationship between the enzyme and the substrate. It's the ratio of them breaking apart to them sticking together.

  • A low Ks means the enzyme and substrate bind tightly (they are "clingy").
  • A high Ks means they bind loosely and fall apart easily.
Ks = ([E] * [S]) / [ES]

Step 6: Putting It All Together (The Derivation)

Now we will combine everything to create one master equation.

  1. Start with the rearranged Ks equation:
    [ES] = ([E] * [S]) / Ks
  2. Substitute the formula for free enzyme [E] from Step 3:
    [ES] = (([E]total - [ES]) * [S]) / Ks
  3. Do some algebra to solve for [ES]:

    Ks * [ES] = ([E]total * [S]) - ([ES] * [S])

    Ks * [ES] + [ES] * [S] = [E]total * [S] [ES] * (Ks + [S]) = [E]total * [S]

    [ES] = ([E]total * [S]) / (Ks + [S])

  4. Substitute this new expression for [ES] back into our speed equation from Step 2 (V = k_p * [ES]):
    V = k_p * (([E]total * [S]) / (Ks + [S]))
  5. Finally, substitute Vmax for k_p * [E]total (from Step 4):
    V = (Vmax * [S]) / (Ks + [S])

And there it is! The famous equation.

The Michaelis-Menten Equation

As noted, under steady-state conditions, Ks is often written as Km (the Michaelis Constant). They represent the same concept of binding affinity.

V = (Vmax * [S]) / (Km + [S])

This equation perfectly describes how the reaction speed (V) changes depending on how much substrate ([S]) is available. It is one of the most important formulas in all of biochemistry!

Enzyme Classification

With thousands of known enzymes, a systematic method for naming and classifying them is essential. The International Union of Biochemistry and Molecular Biology (IUBMB) established a classification system based on the type of reaction catalyzed.

Every enzyme is assigned a unique EC number (Enzyme Commission number), which consists of four numbers separated by dots (e.g., EC 2.7.1.1).

  • The first number indicates the main class of reaction.
  • The second and third numbers denote subclasses and sub-subclasses.
  • The fourth number is the serial number of the enzyme within its sub-subclass.

There are six main classes of enzymes:

1. Oxidoreductases (EC 1):

Catalyze oxidation-reduction (redox) reactions, involving the transfer of electrons or hydrogen atoms.

  • General reaction: A(reduced) + B(oxidized) <=> A(oxidized) + B(reduced)
  • Examples:
    • Dehydrogenases: Remove hydrogen atoms (e.g., lactate dehydrogenase).
    • Oxidases: Catalyze reactions where oxygen is the electron acceptor (e.g., cytochrome c oxidase).
    • Reductases: Catalyze reactions where a substance is reduced.

2. Transferases (EC 2):

Catalyze the transfer of a functional group (e.g., methyl, acetyl, phosphate) from one molecule (donor) to another (acceptor).

  • Key characteristic: Involves two substrates and two products.
  • General reaction: A-X + B <=> A + B-X
  • Examples:
    • Kinases: Transfer phosphate groups, usually from ATP (e.g., hexokinase).
    • Transaminases: Transfer amino groups (e.g., alanine transaminase).

3. Hydrolases (EC 3):

Catalyze the hydrolysis (cleavage) of bonds by the addition of water.

  • Key characteristic: A molecule is broken down into two smaller molecules, with water being consumed.
  • General reaction: A-B + H₂O -> A-H + B-OH
  • Examples:
    • Esterases: Hydrolyze ester bonds (e.g., lipase).
    • Peptidases/Proteases: Hydrolyze peptide bonds in proteins (e.g., trypsin, pepsin).
    • Nucleases: Hydrolyze phosphodiester bonds in nucleic acids.
    • Amylases: Hydrolyze glycosidic bonds in carbohydrates.

4. Lyases (EC 4):

Catalyze the cleavage of C-C, C-O, C-N, or other bonds by mechanisms other than hydrolysis or oxidation, often forming double bonds or rings. They can also catalyze the reverse reaction.

  • Key characteristic: No water is involved in the bond breaking. Often produce a product with a double bond.
  • General reaction: A-B -> X + Y (where X and Y often include a double bond) or vice versa.
  • Examples:
    • Decarboxylases: Remove carbon dioxide (e.g., pyruvate decarboxylase).
    • Aldolases: Cleave C-C bonds (e.g., fructose-1,6-bisphosphate aldolase).
    • Fumarase: Adds water across a double bond.

5. Isomerases (EC 5):

Catalyze the rearrangement of atoms within a single molecule, resulting in an isomer.

  • Key characteristic: Only one substrate and one product.
  • General reaction: A <=> isomer of A
  • Examples:
    • Racemases/Epimerases: Change the stereochemistry around a single chiral center.
    • Mutases: Catalyze the shift of a functional group from one position to another within the same molecule (e.g., phosphoglycerate mutase).

6. Ligases (EC 6):

Catalyze the joining of two molecules (ligation) with the concomitant hydrolysis of a high-energy pyrophosphate bond in ATP or a similar nucleoside triphosphate. These are "synthesis" enzymes.

  • Key characteristic: Requires energy input, usually from ATP.
  • General reaction: A + B + ATP -> A-B + ADP + Pi (or AMP + PPi)
  • Examples:
    • Synthetases: Form new bonds (e.g., DNA ligase, glutamine synthetase).
    • Carboxylases: Add a carboxyl group (e.g., pyruvate carboxylase).

Over The Hill

Oxidoreductases, Transferases, Hydrolases

Like I Like

Lyases, Isomerases, Ligases

Enzyme Inhibition

Enzyme inhibition is a process by which molecules (inhibitors) bind to enzymes and decrease their activity. This is a vital mechanism for regulating metabolic pathways and forms the basis for the action of many drugs, toxins, and pesticides.

Enzyme inhibitors can be classified based on two main criteria:

  • Reversibility: Whether the inhibitor forms a transient or permanent bond with the enzyme.
  • Mechanism of Action: How the inhibitor interacts with the enzyme-substrate binding or catalysis.

A. Reversible Inhibition

Reversible inhibitors bind to enzymes via non-covalent bonds (e.g., hydrogen bonds, ionic bonds, hydrophobic interactions). They can dissociate from the enzyme, allowing the enzyme to regain activity. There are three main types:

1. Competitive Inhibition:

  • Mechanism: The inhibitor (I) structurally resembles the natural substrate (S) and competes with the substrate for binding to the active site of the enzyme.
  • E + S <=> ES -> E + P
  • E + I <=> EI (no product formed)
  • Effect on Kinetics:
    • Vmax: Unchanged. At very high substrate concentrations, the substrate can outcompete the inhibitor for the active site, eventually reaching the original Vmax.
    • Km: Appears to increase. More substrate is required to achieve half Vmax because the inhibitor reduces the effective enzyme concentration available for substrate binding. The enzyme's apparent affinity for the substrate decreases.
  • Example: Malonate is a competitive inhibitor of succinate dehydrogenase (an enzyme in the Krebs cycle), competing with its substrate, succinate. Many sulfa drugs are competitive inhibitors of bacterial enzymes.

2. Non-Competitive Inhibition (or Mixed Non-Competitive Inhibition):

  • Mechanism: The inhibitor binds to a site on the enzyme other than the active site (an allosteric site). This binding causes a conformational change that reduces the enzyme's catalytic efficiency. The inhibitor can bind to either the free enzyme (E) or the enzyme-substrate complex (ES).
  • E + S <=> ES -> E + P
  • E + I <=> EI (inactive)
  • ES + I <=> ESI (inactive)
  • Effect on Kinetics:
    • Vmax: Decreases. The inhibitor effectively reduces the concentration of active enzyme, leading to a lower maximum reaction rate, regardless of substrate concentration.
    • Km: Unchanged (in "pure" non-competitive inhibition). The inhibitor does not directly affect the enzyme's affinity for substrate binding at the active site.
  • Example: Many heavy metal ions (e.g., lead, mercury) act as non-competitive inhibitors by binding to sulfhydryl groups on enzymes.

3. Uncompetitive Inhibition:

  • Mechanism: The inhibitor binds only to the enzyme-substrate (ES) complex, not to the free enzyme. This binding creates an ESI complex that cannot proceed to form a product.
  • E + S <=> ES -> E + P
  • ES + I <=> ESI (inactive)
  • Effect on Kinetics:
    • Vmax: Decreases. The formation of the ESI complex reduces the concentration of functional ES complex, thus lowering the maximum rate.
    • Km: Appears to decrease. The inhibitor binding to ES "pulls" the ES equilibrium towards its formation, making the enzyme appear to have a higher affinity for the substrate.
  • Example: Lithium is an uncompetitive inhibitor of inositol monophosphatase.

B. Irreversible Inhibition

Irreversible inhibitors form a stable, covalent bond with the enzyme, or very tightly-bound non-covalent interactions that are effectively permanent. They permanently inactivate the enzyme.

  • Mechanism: The inhibitor typically binds to or reacts with an amino acid residue in or near the active site, destroying its catalytic or binding ability.
  • Effect: Reduces the concentration of active enzyme permanently. The enzyme cannot regain activity.
  • Example:
    • Organophosphates: Irreversibly inhibit acetylcholinesterase, a critical enzyme for nerve function.
    • Aspirin: Irreversibly inhibits cyclooxygenase (COX) enzymes by acetylating a serine residue in the active site.
    • Penicillin: Irreversibly inhibits transpeptidases involved in bacterial cell wall synthesis.

C. Allosteric Regulation (A form of non-competitive interaction)

While not strictly an "inhibition" type in the same kinetic sense, allosteric regulation is a crucial mechanism of enzyme control.

  • Mechanism: Allosteric effectors (modulators) bind to a site other than the active site (the allosteric site), causing a conformational change that affects the active site's activity.
  • Allosteric inhibition decreases enzyme activity.
  • Allosteric activation increases enzyme activity.
  • Key characteristic: Allosteric enzymes often display sigmoidal (S-shaped) kinetics rather than hyperbolic Michaelis-Menten kinetics.
  • Example: Feedback inhibition, where the end product of a metabolic pathway inhibits an enzyme early in the pathway (e.g., ATP inhibits phosphofructokinase in glycolysis).

Enzymes in Medicine

Enzymes are invaluable tools and indicators in modern medicine, playing a crucial role in diagnosis, therapy, and laboratory assays. Their specificity and catalytic power make them uniquely valuable.

1. Enzymes as Diagnostic Indicators/Markers (Enzyme Diagnostics)

The activities of specific enzymes in bodily fluids, particularly plasma (blood serum), serve as vital diagnostic indicators for various diseases. While enzymes function intracellularly, their presence and elevated levels in the plasma typically signal tissue damage or cellular dysfunction.

Mechanism:

  • Healthy State: In healthy individuals, intracellular enzyme levels in plasma are low and relatively constant, establishing "reference values."
  • Disease State: Tissue damage (e.g., due to anoxia, infection, or necrosis) leads to increased release of intracellular enzymes from damaged cells into the bloodstream.
  • Physiological Elevations: It's important to note that some physiological events, like intense skeletal muscle exertion, can also cause transient, non-pathological elevations of certain enzymes.

Causes of Altered Plasma Enzyme Levels:

  • Increased Values (Elevated Activity):
    • Increased Cell Membrane Permeability: Early cell damage can lead to leakage of cytosolic enzymes (e.g., ALT, LDH, CK).
    • Cell Necrosis (Cell Death): More severe damage results in the release of membrane-bound and mitochondrial enzymes (e.g., ALP, AST).
    • Induction of Enzyme Synthesis: Certain drugs can increase the synthesis and subsequent release of enzymes.
  • Decreased Values:
    • Inhibition of Activity: Some drugs can directly inhibit enzyme activity.
    • Inhibition of Synthesis: Severe cell damage or certain drugs can reduce the rate of enzyme production.

Isoenzymes as Specific Indicators:

  • Concept: Many enzymes exist in different forms called isoenzymes (or isozymes), which catalyze the same reaction but differ slightly in their amino acid sequence and physical properties.
  • Diagnostic Value: The specific pattern of isoenzymes in plasma can pinpoint the exact tissue or organ that has been damaged.

Commonly Assayed Enzymes for Diagnosis (Examples):

Enzyme Primary Location Cause of Elevated Plasma Level
Acid Phosphatase (ACP)ProstateProstatic cancer
Alkaline Phosphatase (ALP)Bone, LiverRickets, obstructive jaundice, cancer of bone/liver
Alanine Aminotransferase (ALT)Liver (also muscle, heart)Hepatitis, jaundice (highly specific for liver damage)
Aspartate Aminotransferase (AST)Heart, Muscle, LiverMyocardial infarction, muscle damage, hepatitis
Amylase (AM)PancreasAcute pancreatitis, peptic ulcer
gamma-Glutamyl Transferase (GMT)Liver, Kidney, PancreasHepatitis, alcoholic liver damage, cholestasis

Commonly Assayed Isoenzymes (Examples):

Isoenzyme Primary Location Cause of Elevated Plasma Level
Creatine Kinase (CK) isoenzymes:
CK-MBHeartMyocardial infarction (heart attack)
CK-MMSkeletal muscleMuscular dystrophy, muscle trauma
Lactate Dehydrogenase (LDH) isoenzymes:
LDH1 (>LDH2)Heart, Kidney, RBCsMyocardial infarction, kidney disease, megaloblastic anemia
LDH5Liver, MuscleLiver disease, muscle damage

2. Enzymes as Therapeutic Agents (Enzymotherapy)

Enzymes are used directly as drugs to treat a variety of conditions.

Applications:

  • Substitution Therapy: Replacing missing digestive enzymes (e.g., pepsin, trypsin, lipase) in conditions like pancreatic insufficiency using preparations like Pancreatin.
  • Debridement/Tissue Clearance: Using enzymes like proteinases and collagenase to remove dead tissue or fibrin deposits to aid wound healing.
  • Fibrinolysis (Clot Busting): Using enzymes such as streptokinase and urokinase to activate plasminogen, which then breaks down fibrin clots in conditions like pulmonary embolism or myocardial infarction.
  • Anti-inflammatory and Immunomodulatory Effects: Orally administered enzyme mixtures (e.g., Wobenzyme) are used for inflammatory diseases, autoimmune diseases, and even cancer support by interacting with plasma proteins and modulating immune responses.
  • Specific Disease Treatment:
    • Asparaginase and Glutaminase: Used in some leukemias to deplete essential amino acids needed by cancer cells.
    • Hyaluronidase: Can be used in heart attack to help degrade hyaluronic acid, reducing tissue swelling.
    • Lysozyme: Has antibiotic action by hydrolyzing bacterial cell walls.
    • Rhodanase: Used in cyanide poisoning to convert toxic cyanide to less toxic thiocyanate.
    • beta-Lactamase: Used to treat penicillin allergy by degrading penicillin.
    • Uricase: Used in gout to convert insoluble urate to more soluble allantoin.

3. Enzymes as Diagnostic Tools (in Clinical Laboratory Assays)

Enzymes are used as highly specific reagents in laboratory tests to measure the concentration of various substances in biological samples.

Advantages over Chemical Methods:

  • High Specificity: Enzyme methods are generally much more specific than traditional chemical methods, leading to more accurate results.

Applications and Examples:

  • Commercial Kits and Diagnostic Strips: Enzymes are incorporated into readily available kits and strips for testing.
  • Glucose Determination: Glucose oxidase and peroxidase are used to measure glucose levels in blood or urine for diabetic monitoring.
  • Cholesterol Determination: Cholesterol esterase and cholesterol oxidase are used to measure total cholesterol.
  • Urea Determination: Urease is used to break down urea, and the products are then measured.
  • Immunochemical Analysis (e.g., ELISA): Enzymes like peroxidase and alkaline phosphatase are conjugated to antibodies. The enzyme then catalyzes a color-forming reaction, allowing for the sensitive detection and quantification of antigens or antibodies in a sample.

Isoenzymes: Multiple Forms, Specific Diagnosis

Isoenzymes (or isozymes) are different molecular forms of the same enzyme that catalyze the same biochemical reaction. While their catalytic function is identical, they possess different chemical and physical properties due to slight variations in their amino acid sequence and/or composition.

Key Differentiating Properties of Isoenzymes:

  • Electrophoretic Mobility: They migrate differently in an electric field due to variations in charge.
  • Kinetic Properties: May exhibit different Km values (affinity for substrate) or Vmax (maximum reaction rate).
  • Amino Acid Sequence and Composition: Subtle differences in primary structure.
  • Heat Stability: Some isoenzymes are more stable at higher temperatures than others.
  • Sensitivity to Inhibitors: May respond differently to various inhibitors.

The ability to distinguish between isoenzymes is of paramount importance in clinical diagnostics, as it allows for the pinpointing of specific tissue damage.

1. Lactate Dehydrogenase (LDH)

General Information:

  • Function: LDH is an oxidoreductase (EC 1.1.1.27) that reversibly catalyzes the interconversion of lactate and pyruvate, using NAD⁺/NADH as a coenzyme.
  • Normal Values:
    • Serum: 100-200 U/L
    • CSF (Cerebrospinal Fluid): 7-30 U/L
    • Urine: 40-100 U/L

Clinical Significance of LDH (in Plasma/Serum):

  • Normal Serum: LDH2 is usually the predominant isoenzyme; LDH5 is low or absent.
  • Myocardial Infarction (MI): LDH1 levels become greater than LDH2 (LDH1 > LDH2 "flip"), a classic indicator after an MI.
  • Megaloblastic Anemia: Very high elevations of LDH1 and LDH2 (up to 50 times the upper limit).
  • Muscular Dystrophy: Increased LDH5.
  • Toxic Hepatitis/Liver Damage: Significant increase in LDH5 (up to 10 times or more).
  • Renal Disease: Tubular necrosis or pyelonephritis can elevate LDH isoenzymes.
  • Neoplastic Diseases (Cancers): Total LDH often increases.
    • LDH5: Increased in breast cancer, CNS malignancies, prostatic carcinoma.
    • LDH2 & LDH3: Increased in leukemias.

CSF Analysis:

  • Bacterial Meningitis: Elevated LDH4 and LDH5.
  • Viral Meningitis: Elevated LDH1.
  • Metastatic Tumors: Elevated LDH5.
  • Neonatal Intracranial Hemorrhage: Elevated LDH levels are associated with seizures and hydrocephalus.

2. Creatine Kinase (CK) / Creatine Phosphokinase (CPK)

General Information:

  • Function: CK catalyzes the reversible phosphorylation of creatine using ATP to form creatine phosphate and ADP. This is crucial for providing an immediate source of ATP in rapidly contracting muscle.
  • CK Isoenzymes (3 Forms): The two types of subunits (B and M) combine to form three dimeric isoenzymes.
  • Normal Serum Values:
    • Males: 15-100 U/L
    • Females: 10-80 U/L

Clinical Significance of CK (in Plasma/Serum):

  • Myocardial Infarction (MI):
    • CK-MB is the most specific indicator for MI.
    • Levels increase within 4-6 hours post-MI, peak at 18-24 hours, and return to normal within 2-3 days.
    • Even though CK-MB is a small percentage of total CK, its elevation is highly indicative of heart damage.
  • Muscle Diseases/Injury:
    • Muscular Dystrophy: Markedly elevated total CK levels, primarily due to CK-MM leakage.
    • Crush Injury, Fractures: Significant elevations in total CK, reflecting muscle or brain tissue damage.

Atypical Forms of CK:

These forms can interfere with the interpretation of standard CK isoenzyme assays.

  • Macro-CK (CK-macro):
    • Formation: An aggregated form, typically CK-BB complexed with IgG, or CK-MM complexed with lipoproteins.
    • Electrophoretic Migration: Migrates between CK-MB and CK-MM.
    • Clinical Relevance: May indicate benign conditions but can be associated with autoimmune diseases or malignancies.
  • Mitochondrial CK (CK-Mi):
    • Formation: A distinct isoenzyme found bound to the inner mitochondrial membrane.
    • Electrophoretic Migration: Migrates towards the cathode, behind the CK-MM band.
    • Clinical Relevance: Not present in normal serum. Its presence indicates extensive tissue damage and breakdown of mitochondrial membranes, often seen in severe cellular injury.
Biochemistry: Enzymes Exam
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Biochemistry: Enzymes Exam

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