Enzyme Characteristics and Classification

Enzyme Characteristics

• Enzymes are biological catalysts that accelerate the rate of biochemical reactions without being permanently altered.
• Almost all enzymes are proteins, however, some nucleic acids can act as enzymes.
• Enzymes exhibit a high degree of specificity, meaning they are sensitive to small changes in the chemical structure of substrates.
• Enzyme activity is heavily influenced by factors such as temperature, pH, substrate concentration, cofactor availability, and the ionic environment.

Classification of Enzymes

• Enzymes are categorized into six major classes based on the type of reaction they catalyze:
  • Oxidoreductases: Catalyze oxidation-reduction reactions.
  • Transferases: Transfer functional groups between molecules.
  • Hydrolases: Break down molecules by adding water.
  • Lyases: Cleave molecules without using water or oxidation.
  • Isomerases: Rearrange atoms within a molecule.
  • Ligases: Join two molecules together using energy from ATP.

The Active Site of an Enzyme

• The active site is a small region on the enzyme's surface where substrate binding and catalysis occur.
• Substrate binding involves weak interactions between the substrate and amino acid side chains within the active site.
• Enzyme specificity arises from the geometric and chemical complementarities between the substrate and the active site.
• Interactions include ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.
• Catalysis is achieved by altering the reaction pathway and transition state energy through interactions with active site amino acids.
• These interactions can involve acid-base catalysis, nucleophilic and electrophilic interactions, hydrophobic effects, ionic stabilization, and covalent bond formation between substrate and enzyme.

Substrate Binding Hypotheses

• The lock and key hypothesis proposes that the active site is a pre-existing, exact complement to the substrate.
• The induced fit hypothesis suggests that the active site changes shape upon substrate binding to achieve a precise fit.

Enzyme Cofactors

• Cofactors are non-protein molecules essential for enzyme activity.
• Some cofactors are metal ions, while others are organic molecules called coenzymes.
• Coenzymes often derive from vitamins, and their deficiency can lead to metabolic disorders.

Nicotinamide Adenine Dinucleotide (NAD+)

• NAD+ is a coenzyme involved in various oxidation-reduction reactions.
• It can accept two electrons and one proton in its oxidized form (NAD+) or donate them in its reduced form (NADH).
• NAD+ is primarily used as an electron acceptor in catabolic reactions and as an electron donor in oxidative phosphorylation.
• Its structure consists of adenine, ribose, phosphates, and a nicotinamide ring.
• The nicotinamide ring is the site of catalytic activity, accepting or donating electrons and protons.
• Deficiency in nicotinamide (vitamin B3) can lead to pellagra.

Nicotinamide Adenine Dinucleotide Phosphate (NADP+)

• NADP+ is structurally similar to NAD+ but has an extra phosphate group on a ribose ring.
• It functions in oxidation-reduction reactions but is primarily used as a reducing agent (NADPH) in anabolic reactions.

Thermodynamics

• Standard free energy change (ΔGo) measures the energy released or consumed by a reaction under standard conditions.
• Standard free energy change is calculated using the equilibrium constant (K'eq): ΔGo' = -RTlnK'eq.
• A negative ΔGo' indicates that the reaction favors product formation at equilibrium.
• A ΔGo' of zero indicates equal amounts of substrate and product at equilibrium.
• A positive ΔGo' indicates that the reaction favors reactant formation at equilibrium.
• Enzymes do not alter the standard free energy change of a reaction.
• The standard free energy change does not provide information about reaction rates, only the direction of the reaction at equilibrium.

Enzyme Characteristics

• Enzymes are biological catalysts that accelerate biochemical reactions without being permanently altered.
• Most enzymes are proteins, but some RNA molecules can also act as enzymes.
• Enzymes exhibit high specificity for their substrates, meaning they can distinguish between very similar molecules.
• Enzyme activity is influenced by factors such as temperature, pH, substrate concentration, cofactor availability, and ionic environment.

Classification of Enzymes

• Enzymes are categorized into six major classes based on the type of reaction they catalyze:
  • Oxidoreductases: Catalyze oxidation-reduction reactions.
  • Transferases: Transfer functional groups between molecules.
  • Hydrolases: Break down molecules by adding water.
  • Lyases: Cleave chemical bonds without adding water.
  • Isomerases: Rearrange atoms within a molecule, creating isomers.
  • Ligases: Join two molecules together, often using energy from ATP.

Enzyme Active Site

• The active site is a small region on the enzyme's surface where substrate binding and catalysis occur.
• It is composed of amino acid side chains that interact with the substrate through a combination of weak forces:
  • Ionic bonds
  • Hydrogen bonds
  • Hydrophobic interactions
  • van der Waals forces
• The active site's shape and chemical properties determine the enzyme's specificity for its substrate..

Substrate Binding Models

• Lock-and-key hypothesis: The enzyme active site is a perfect fit for the substrate, like a key fitting into a lock.
• Induced fit hypothesis: The enzyme's active site changes shape upon substrate binding, leading to a more precise fit.

Coenzymes

• Coenzymes are non-protein organic molecules that are required for the activity of certain enzymes.
• They often carry electrons or functional groups during enzymatic reactions.
• Examples of important coenzymes include:
  • NAD+ and NADP+: Serve as electron carriers in oxidation-reduction reactions.
  • FAD: Acts as an electron carrier in oxidation-reduction reactions.
  • Coenzyme A: Important in metabolic pathways, particularly those involving acyl group transfers.

Enzyme Kinetics

• Enzymes affect reaction rates, but they do not change the reaction equilibrium.
• The equilibrium constant (K) of a reaction is a measure of the relative amounts of substrates and products at equilibrium.
• Enzymes cannot change the equilibrium constant, as it is determined by the energy levels of the reactants and products.

Thermodynamics of Enzyme Catalysis

• Free Energy (ΔG): The amount of energy released or consumed by a reaction.
  • A negative ΔG indicates a favorable reaction, where energy is released.
  • A positive ΔG indicates an unfavorable reaction, requiring energy input.
• Standard Free Energy (ΔGo'): The free energy change under standard conditions, where all reactants and products are present at 1 M concentration and pH 7.
• Relationship between ΔGo' and equilibrium constant (K'eq):
  • ΔGo' = -RTlnK'eq
• Key Points about ΔGo':
  • A negative ΔGo' favors product formation at equilibrium.
  • A ΔGo' of zero indicates equal amounts of substrate and product at equilibrium.
  • A positive ΔGo' favors reactant formation at equilibrium.
  • Enzymes do not change the ΔGo' of a reaction.
  • The ΔGo' of a reaction does not provide information about reaction rates.

Enzyme-Substrate Interactions

• Enzymes undergo conformational changes in their active sites upon substrate binding.
• Hexokinase provides an example of induced fit: the active site changes significantly when glucose binds, closing around the substrate.

Effect of Temperature on Enzyme Activity

• Enzyme activity is highly sensitive to temperature changes.
• Enzyme activity increases as temperature rises, up to an optimal temperature range.
• Above a certain critical temperature, enzymes denature and lose activity irreversibly.

Effect of pH on Enzyme Activity

• Enzymes are sensitive to changes in pH.
• Each enzyme has an optimal pH range for maximum activity.
• Enzyme activity decreases and enzymes can be denatured at pH extremes.

Enzyme Catalytic Activity

• Enzyme activity is measured as µmole of product/minute (Enzyme Units).
• Specific activity measures the ratio of enzyme activity to protein content (Enzyme Units/mg of protein).
• Specific activity increases with enzyme purification and indicates homogeneity when it plateaus.

Enzyme Turnover Number

• Turnover number (Kcat) represents the substrate molecules converted per second per enzyme molecule.
• It is an intrinsic property of the enzyme, unlike specific activity, and does not change with purification.
• Turnover numbers vary widely, from less than 1 to over 40,000,000 per second.

Rate Enhancements by Enzymes

• Enzyme catalysis drastically increases reaction rates compared to uncatalyzed reactions.
• Rate enhancements can range from 10⁶ to 10¹⁷.
• For example, OMP decarboxylase accelerates a reaction from millions of years to seconds.

Cofactor Molecules

• Many enzymes require cofactors (coenzymes) for activity.
• Cofactors can be inorganic ions or organic molecules.
• Enzymes lacking essential cofactors are called apoenzymes, while those with cofactors bound are holoenzymes.

ATP as a Cofactor

• ATP is a nucleotide cofactor containing adenine, ribose, and three phosphate groups.
• Hydrolysis of phosphate bonds in ATP releases energy, particularly for the γ and β phosphates.
• ATP hydrolysis powers various biochemical reactions and can be coupled to energy-requiring processes.

FAD and FMN as Cofactors

• FAD and FMN are cofactors involved in oxidation-reduction reactions.
• Both contain an isoalloxazine ring essential for catalysis.
• They differ in their recognition by specific enzymes.

Enzyme-Catalyzed Reaction Pathway

• The simplest kinetic scheme involves enzyme (E) binding substrate (S) to form an enzyme-substrate complex (ES).
• A reaction occurs within the complex, yielding product (P) which is then released.
• The enzyme is then free to participate in another catalytic cycle.

Enzyme Effect on Reaction Equilibrium

• Enzymes accelerate the forward and reverse reactions to the same extent, not changing reaction equilibrium.
• ΔG (free energy) of a reaction determines its thermodynamic favorability.
• Enzymes affect reaction rates, but not the relative energy levels of substrates and products.

Induced Fit Model

• Enzymes undergo conformational changes in their active site when a substrate binds.
• The degree of change varies between different enzymes.
• Hexokinase demonstrates this with a significant conformational change in its active site upon glucose binding.
• In the absence of glucose, the active site is an open cleft.
• When glucose binds, the cleft closes and envelopes the substrate.

Effect of Temperature on Enzyme Activity

• Enzyme activity is sensitive to temperature changes.
• At lower temperatures, enzyme activity increases as temperature rises.
• This relationship is measured by Q10, the increase in activity for every 10-degree rise in temperature.
• Q10 for enzyme reactions is generally higher than for chemical reactions.
• At higher temperatures, enzyme activity increases to an optimal range and then decreases.
• Above a critical temperature, enzymes are denatured and lose activity irreversibly.

Effect of pH on Enzyme Activity

• Enzymes are sensitive to pH fluctuations.
• There is an optimal pH range for each enzyme where maximum activity is observed.
• Activity decreases at pH values outside this range.
• Extreme pH values can denature most enzymes.
• pH influences both the substrate and the active site's shape and charge, affecting enzyme functionality in catalysis.

Enzyme Specific Activity

• Catalytic activity describes an enzyme's ability to convert substrate (S) to product (P).
• It's typically measured as µmole of product/minute (Enzyme Units).
• Specific activity is the ratio of enzyme activity to the amount of protein present (Enzyme Units/mg of protein).
• Specific activity is generally low in crude biological extracts and increases as the enzyme is purified.
• Reaching homogeneity, where further purification doesn't increase specific activity, indicates a pure enzyme preparation.

Enzyme Turnover Number

• The turnover number (Kcat) indicates the molecules of substrate converted per second per enzyme molecule (or active site for multi-subunit enzymes).
• Kcat is calculated using the formula: Kcat = Vmax/[Et].
• Unlike specific activity, the turnover number is an intrinsic property of an enzyme and doesn't change with purification.

Cofactor Molecules

• Many enzyme reactions require cofactors (coenzymes) in addition to the protein structure for catalysis.
• Cofactors can be inorganic ions or organic molecules.
• An enzyme lacking an essential cofactor is an apoenzyme.
• The enzyme with the cofactor bound is a holoenzyme.
• Organic cofactors participate in oxidation-reduction reactions and the transfer of organic groups.
• Many organic cofactors are vitamins or contain vitamin components.
• Inorganic cofactors participate in oxidation-reduction reactions, substrate binding, and maintain the correct protein conformation.

ATP as a Cofactor

• Adenosine triphosphate (ATP) is a nucleotide cofactor containing an adenine base, ribose sugar, and three phosphate groups.
• ATP hydrolysis releases energy through breaking bonds between two phosphate groups.
• This hydrolysis releases energy for biochemical reactions.

Flavine Adenine Dinucleotide (FAD)

• FAD is a coenzyme containing adenosine diphosphate connected to ribitol and an isoalloxazine ring system.
• Riboflavin, a B vitamin, is a component of FAD.
• The isoalloxazine ring is involved in catalysis.

Flavine Mononucleotide (FMN)

• FMN is a coenzyme containing phosphate connected to ribitol and an isoalloxazine ring system.
• It's the isoalloxazine ring that contributes to catalysis.
• FMN functions similarly to FAD but is recognized by different enzymes.

The Pathway of an Enzyme Catalyzed Reaction

• Enzymes (E) bind to their substrates (S) to form an enzyme-substrate complex (ES).
• A biochemical reaction occurs within the ES complex, converting the substrate to a product (P).
• The product is then released, and the enzyme is free to engage in another catalytic cycle.

Standard Free Energy

• Standard free energy (ΔGo) measures the energy released or consumed by a reaction with all substrates at 1 M concentrations under standard conditions.
• ΔGo is useful for chemists and physicists but less relevant for biochemists as it doesn't consider biological pH values (around pH 7.0).

Standard Free Energy for Biologists

• Standard free energy (ΔGo') is a modified version for biologists that accounts for a hydrogen ion concentration at pH 7.0 (10-7 M).
• ΔGo' is denoted by a prime (') to distinguish it from the standard free energy (ΔGo).

The Relationship Between Standard Free Energy and Equilibrium Constant

• At equilibrium, ΔG equals zero.
• There's a mathematical relationship: ΔGo' = -RTlnK'eq, where K'eq is the equilibrium constant.
• This means the equilibrium constant is linked to the standard free energy and is not altered by the presence of an enzyme.

Facts Concerning Standard Free Energy

• A reaction with a negative ΔGo' favors product formation at equilibrium.
• A reaction with a ΔGo' of zero yields equal amounts of substrate and product at equilibrium.
• A reaction with a positive ΔGo' favors reactant formation at equilibrium.
• Enzymes do not change the ΔGo' of a reaction.
• The ΔGo' of a reaction only indicates the direction of the reaction at equilibrium and doesn't reveal anything about the reaction rate.

Enzyme Characteristics

• Enzymes are biological catalysts. They speed up reactions without being consumed in the process.
• Most enzymes are proteins; however, some nucleic acids can act as enzymes.
• Enzymes exhibit high specificity, often responding strongly to small changes in substrate structure.
• Enzyme activity can be significantly impacted by factors like temperature, pH, substrate concentration, cofactors, and ionic environment.

Enzyme Classification

• There are six major categories of enzymes, each performing specific reactions.
• Oxidoreductases: These enzymes catalyze oxidation-reduction reactions, involving the transfer of electrons.
• Transferases: Transfer functional groups (like methyl, acyl, phosphate) between molecules.
• Hydrolases: Break down molecules by adding water, like during digestion.
• Lyases: Break chemical bonds, often leaving double bonds or rings.
• Isomerases: Rearrange atoms within a molecule, creating isomers.
• Ligases: Join two molecules together; require energy input, often from ATP.

Active Site of an Enzyme

• The active site is a specific region on an enzyme where substrate binds and catalysis occurs.
• It's a small area on the surface comprised of particular amino acid side chains.
• Binding: The substrate interacts with the active site through weak forces: ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.
• Specificity: The shape and chemical properties of the active site determine the substrate that can bind.
• Catalysis: Specific amino acid side chains within the active site interact with the substrate, altering the reaction pathway and lowering the activation energy.

Lock & Key Hypothesis

• This model proposes that the active site has a fixed shape that perfectly complements the substrate.
• The substrate fits into the active site like a key into a lock.

Induced Fit Hypothesis

• This model suggests that the active site is flexible and adjusts its shape to fit the substrate precisely.
• The interaction with the substrate induces a conformational change in the enzyme, creating a better fit.

NAD+ and NADP+

• NAD+ and NADP+ are common coenzymes involved in many oxidation-reduction reactions.
• NAD+ is a dinucleotide with a nicotinamide ring, which can be reduced to NADH.
• NADP+ is structurally similar to NAD+, but has an extra phosphate group.
• Both molecules can accept or donate electrons, but NADP+ is generally used in its reduced form (NADPH) as an electron donor in anabolic reactions.

Thermodynamics of Enzyme Activity

• Enzymes don't change the thermodynamics of a reaction; they don't alter the change in free energy (ΔG) or the equilibrium constant (K'eq).
• The ΔG determines whether a reaction is favorable (negative ΔG) or unfavorable (positive ΔG).
• Enzymes do affect the rate of a reaction by lowering the activation energy (Ea).
• A lower activation energy means a reaction proceeds faster.

Standard Free Energy

• Standard free energy (ΔG°) measures energy change under standard conditions: all reactants at 1 M concentration, 298K (25°C), 1 atm pressure.
• Standard free energy for biological systems (ΔG°') is adjusted for pH 7.0 ([H+] = 10^-7 M).

Relationship Between ΔG°' and Equilibrium Constant

• They are directly related through the equation: ΔG°' = -RTlnK'eq
• K'eq is the equilibrium constant, indicating the ratio of products to reactants at equilibrium.
• A negative ΔG°' indicates a favorable reaction with a high K'eq.
• A positive ΔG°' indicates an unfavorable reaction with a low K'eq.

Enzyme Action Recap

• Enzymes speed up reactions by lowering the activation energy, but they do not change the total free energy change for the reaction.

This quiz covers key features and classifications of enzymes, highlighting their role as biological catalysts. Understand enzyme specificity, factors influencing enzyme activity, and the six major classes of enzymes. Test your knowledge of how enzymes operate and their importance in biochemical reactions.