L19

Enzyme Catalysis and Function

Introduction

• Enzymes are crucial for cell metabolism and controlling cell functions.
• This lecture builds on the importance of protein shape discussed previously, as enzyme shape is critical for function.

Learning Goals

• Understand that enzymes lower the activation energy of a reaction.
• Understand that the substrate must have a matching shape to fit the enzyme's active site.
• Learn about the lock and key and induced fit models of enzyme-substrate binding.
• Understand that enzymes often require cofactors to be fully active.
• Understand how cofactors enhance enzyme activity.
• Learn about feedback inhibition and other mechanisms of enzyme regulation.

Enzymes and Cell Metabolism

• Chemical reactions in cells occur at lower temperatures and reactant concentrations than they would normally.
• Enzymes accelerate these reactions under physiological conditions.
• Reactions can occur without enzymes, but much more slowly.
• Enzymes are catalysts that accelerate the conversion of substrate to product without being changed themselves.
• Most enzymes are proteins; ribozymes (RNA enzymes) are a rare exception and will not be the focus of this module.

Enzyme Specificity

• Enzymes perform highly specific reactions, catalyzing one reaction or binding to multiple substrates for the same reaction type.
• Specificity arises from the shape of the enzyme, particularly the active site.
• The active site is the region of the enzyme that performs the activity.
• Enzyme shape determines substrate specificity and the type of reaction catalyzed.

Cellular Metabolism

• Catalysis allows cells to control metabolism through a balance of catabolic and anabolic pathways.
• Catabolism (Catabolic Pathways):
  • Breaking down food into smaller molecules to generate energy.
• Anabolism (Anabolic Pathways):
  • Using energy from catabolism to synthesize new molecules from these building blocks.
  • Remember: Anabolic steroids build up muscle.
• Cellular metabolism involves a fine balance between catabolic and anabolic pathways.

Enzymes and Thermodynamics

• Enzymes can only drive reactions that would occur spontaneously; they follow the laws of thermodynamics.
• Energy cannot be created or destroyed.
• Free Energy (ΔG):
  • The energy that drives reactions.
  • Spontaneous reactions lead to a loss of free energy (negative $\Delta G$).
• Activation Energy:
  • The input of energy required to kick-start a reaction.
  • Enzymes work by lowering activation energy, increasing the probability of the reaction occurring.
  • If the amount of activation energy is high then the probability of reaction occurring is low and vice versa.

Transition State

• Substrate conversion to product involves intermediate states.
• Activation energy is required to form the most unstable intermediate state, the transition state.
• In the transition state, the substrate is neither substrate nor product.
• Enzymes facilitate the formation of the transition state, lowering the required activation energy, but they don't change the free energy or reaction equilibrium.

Enzymatic Reactions and Equilibrium

• Enzymatic reactions don't always occur only in the forward direction; the reverse reaction also occurs.
• The initial enzyme-substrate binding isn't all or nothing; dissociation can occur.
• Dissociation constants measure the strength of binding and dissociation rate.
• Enzymes don't change the equilibrium (balance) of the reaction because they lower the activation energy for both the forward and reverse reactions.
• Symbols used to represent enzymatic reaction.
  • E = enzyme
  • S = substrate
  • ES = enzyme-substrate complex

Forward vs. Reverse Reactions

• Substrate to product conversion is more energetically favorable (forward reaction), but product to substrate (reverse reaction) can occur less often because it requires more energy.
• Eventually, an equilibrium is reached where there's no net change in the ratio of substrate to product, and free energy is zero.
• Because the product is often thermodynamically stable, the reverse reaction is often assumed to be irreversible, especially when examining enzyme kinetics (as in the lab class).
• When product is irreversible, the reaction only occurs in the forward direction.

Enzyme-Substrate Binding

• The first step in enzyme catalysis is substrate binding.
• The substrate needs to fit into the shape of the active site for catalysis to occur.
• Enzyme shape determines substrate fit and specificity.
• Even multifunctional enzymes (those that bind multiple substrates) have specificity based on the type of reaction performed.
• Multifunctional enzymes will still bind to one molecule in preference to all others, so they still have a preference.
• All enzymes have a substrate-binding site, with a small region called the catalytic domain or active site.

Regulatory Sites (Allosteric Binding Sites)

• Some enzymes have a regulatory site (also called an allosteric binding site) distant from the substrate-binding site.
• Not all enzymes have them.
• Binding at the regulatory site can change enzyme activity.
  • Allosteric activators: binding causes a shape change that permits substrate binding, increasing activity.
  • Inhibitors: binding changes the shape of the substrate-binding site to prevent binding, inhibiting activity.
• Allosteric means distant.
• The allosteric site causes shape change at the substrate binding site.

Active Site

• The active site is a small cleft or pocket that is part of the substrate binding site.
• It's formed by amino acids/residues from multiple parts of the protein that come together when the protein folds into its tertiary structure.
• Example: Chymotrypsin's active site consists of only three amino acids: Serine 195, Histidine 57, and Aspartic Acid 102, which are not sequential but form a pocket in the tertiary structure.
• Forms a nonpolar microenvironment, keeping out water.

Enzyme-Substrate Interactions

• Weak noncovalent bonds (hydrogen bonds, van der Waals forces, electrostatic attractions) stabilize enzyme-substrate binding.
• Multiple weak interactions must form strongly enough to keep the substrate in place long enough for the enzyme to function.

Lock and Key vs. Induced Fit Models

• Enzymes bind to substrates in one of two ways:
  • Lock and key model.
  • Induced fit model.

Lock and Key Model

• The enzyme and substrate are rigid and perfectly match each other for catalysis to occur.
• The enzyme does not change the shape and substrate doesn't change shape.
• Identified in the late 1800s.
• Specificity comes from the precise arrangement of atoms.

Induced Fit Model

• The enzyme shape doesn't completely match the substrate.
• The substrate binding site is not rigid.
• Binding induces a shape change in the enzyme and sometimes the substrate.
• This modification of shape causes a better fit, stronger binding, and allows catalysis to occur.
• Discovered later (1950s/60s).

Cofactors

• Some enzymes need helpers called cofactors to be fully active.
• Simple enzymes are protein only.
• Some enzymes require a non protein molecule to bind to them and bind to both the active site and regulatory site.
• Cofactors activate the enzyme and allow catalysis to occur.
• Cofactors are non protein and can be either organic or inorganic.
  • They provide chemically versatile functions not found in amino acids.
• Protein + Cofactor = Complex Enzyme.

Apoenzyme vs. Holoenzyme

• Apoenzyme (Apoprotein):
  • The protein component, which is inactive.
• Holoenzyme:
  • The active complex formed when the apoenzyme binds to the cofactor.

Classification of Cofactors

• Cofactors can be classified as:
  • Coenzymes.
  • Prosthetic groups.

Coenzymes vs. Prosthetic Groups

• Coenzymes:
  • Non-protein components that bind to the apoenzyme non-covalently.
• Prosthetic groups:
  • Non-protein components that bind to the apoenzyme tightly through covalent bonds.
• Caution: Some American textbooks refer to all cofactors as coenzymes, which is not technically correct.

How Cofactors Work

1. Stabilize the binding of the substrate.
2. Produce a shape change by binding at the allosteric binding site.
3. Involved in transfer processes by having an organic or inorganic part of a molecule to allow enzyme catalysis to occur.

• Cofactors can bind to the catalytic or regulatory site to stabilize substrate binding or produce allosteric shape changes that increases enzyme activity.
• Some enzymes need multiple cofactors.

Organic vs. Inorganic Cofactors

• Organic cofactors may include heme, vitamin C, nucleotides (NAD).

• Inorganic cofactors may include metal ions or trace elements.

• Enzymes that require metal ions are called metalloenzymes.

• Example: Alcohol dehydrogenase requires both a zinc ion (for substrate binding) and NAD (for a transfer process).

Water-Soluble Vitamins

• Many water-soluble vitamins act as coenzymes and prosthetic groups.
• Cofactors are often derivatives of water-soluble vitamins.

*Clinical Relevance:
The presence of clinical symptoms of dietary enzyme deficiency are caused by enzymes malfunctioning due to not having enough of a vitamin that makes a cofactor in the body.

Optimal Conditions for Enzyme Activity

• Because enzymes are proteins, they need to maintain their correct shape.
• Each enzyme has optimal temperature.
• Each enzyme has optimal pH.
• Each enzyme has optimal ionic conditions that it works under.
• pH affects ionic bonds.
• If the temperature is too low, no energetic collisions.
• Different enzymes will have different optimal pH ranges depending on their function and where they're found within the body.
• Enzymes also have an optimal temperature.

Enzyme Pathways

• Enzymes often function in groups called pathways, involving a series of chemical reactions where the product of one reaction becomes the substrate for the next.
• Can either be metabolic pathways or signaling pathways.
• It is important that enzymes are regulated correctly so that each enzyme functions correctly.

Enzyme Regulation

• Cells regulate enzyme activity in four main ways:

  1. Feedback inhibition.
  2. Changing the number of enzyme molecules.
  3. Changing the location of the enzyme or substrate.
  4. Changing the activity by post-translational modifications or zymogens.

Feedback Inhibition

• The final product of a chemical pathway inhibits an earlier reaction by binding to the allosteric site of the first unique enzyme in the pathway.
  *Feedback inhibition is used to switch off process once there is enough product to prevent waste or prevent accumulation of excess product.
• Malfunctions in feedback inhibition can cause disease.
• Example: Sialuria, where a mutation causes insensitivity to feedback inhibition, leading to a buildup of sialic acid.
  
  
• Sialuria: The production of sugar involved in glycosylation called sialic acid, is regulated by feedback inhibition where one of the products called CMP sialic acid which binds to UDP, acetylglucosamine -two epimerase (at the regulatory site) and switches it off to prevent creation of sialic acid. But among people with Sialuria, they have a mutation where the UDP acetylglucosamine two epimerase is not sensitive to CMP sialic acid and feedback inhibition does not kick in which means that the Sialic acid begins to buildup in the urine.

Changing the Number of Enzyme Molecules

• Cells can increase or decrease the number of enzyme molecules depending on cellular requirements.
• Achieved by:
  • Changing transcription.
  • Changing translation.
    *Changing degradation of the molecules by the proteasome.
• Example: Alcohol dehydrogenase production adjusts based on regular alcohol consumption.

Changing the Location of the Enzyme or Substrate

• Since the first step in enzyme catalysis is enzyme binding to substrate, they must be located together to perform their function correctly.
• Cells can control the location of the substrate.
• Occurs when enzymes have a fixed location.
  *For the location to perform its correct functions there has to be contact with it.
• Example: Glucosal transferases in the Golgi apparatus.

Post-Translational Modifications

*After enzyme has been synthesized it's shape is changed.
*Alter activity by changing shape of enzyme.

• Shape Changes can activate or inhibit of some of the enzymes by revealing substrate binding sites or hiding substrate binding sites.
• Enzymes control these post translational modifications.
• Examples: phosphorylation, glycosylation.

Zymogens

• Inactive enzyme precursors (proenzymes).
• Require irreversible proteolytic cleavage (a bit of them is cut off) to become fully active.
• Example: Blood clotting cascade involves a series of zymogens that are cleaved irreversibly to become activated.

Conclusion

• Enzymes are catalysts and remain unchanged after a catalytic reaction.
• They work by lowering the activation energy, but they don't change the free energy.
• The structure of an enzyme includes a substrate-binding site (with the active site) and sometimes a regulatory site.
• Enzyme binding occurs through the lock and key or induced fit models.
  *All enzymes have optimal conditions and factors influence their functions include: pH, temperature, ionic strength, etc
  *Cofactors can increase enzyme activity and are classified as coenzymes or prosthetic groups.
• Cells regulate enzymes through:
  • Feedback inhibition.
  • Altering enzyme production.
  • Changing enzyme/substrate location.
  • Post-translational modifications or proteolytic cleavages.