Enzymes and Reaction Rates

Enzymes

• Proteins acting as catalysts in chemical reactions.
• Stabilize the transition state and lowers the activation energy through interaction with the substrate.
• Enzyme activity is regulated and can accelerate reaction rates by a million-fold.
• Substrate specific.
• Recycled within the cell for multiple uses.
• Substrates are the reactants in enzyme-catalyzed reactions.
• Rate measures product creation or reactant disappearance over time.

Rate Constant (K)

• K1: Rate constant for enzyme-substrate complex formation from reactants.
• K-1: Rate constant for the substrate being released from the active site.
• K2: Rate constant for product formation.
• K-2: Rate constant for enzyme-substrate complex formation from products.
• Increasing enzyme quantity achieved by increasing gene expression (transcription and translation).

Enzyme Classification

• Oxidoreductases: Catalyze oxidation-reduction reactions, including reductases and dehydrogenases.
• Transferases: Move functional groups between molecules.
  • Kinases phosphorylate by adding a phosphate group from ATP.
• Hydrolases: Cleave bonds with water addition.
• Lyases: Remove or add atoms to create or eliminate double bonds.
• Isomerases: Move functional groups within a molecule.
• Ligases: Join two molecules, requiring ATP.
  • DNA Ligase makes phosphodiester bonds between nucleotides.
• Phosphatases: Dephosphorylate, removing phosphate groups from inorganic molecules.

Cofactors

• Essential for enzyme activity; tightly or covalently bound (Prosthetic Group), e.g., Heme.
• Two classes of cofactors Coenzymes: organic molecules derived from vitamins.
  • Ex: B12; Cyanocobalamin.
• Metals.
• Apoenzyme: Enzyme without its cofactor.
• Holoenzyme: Enzyme with its cofactor.

Thermodynamics

• Gibbs Free Energy (ΔG): The difference in free energy between reactants and products

  • Indicates spontaneity, not the reaction rate.
  • Rate does not change ΔG
  • ΔG = ΔH - TΔS, where ΔH is enthalpy change, ΔS is entropy change, and T is standard temperature, this equation does not account for reactant/product concentrations.

• ΔG Negative: Spontaneous reaction.

• ΔG Zero: Equilibrium.

• ΔG Positive: Nonspontaneous reaction.

Standard Free Energy Change (ΔG°)

• Indicates reactant/product nature, not spontaneity.
• Constant Value.
• ΔG = ΔG° + RTln([C][D]/[A][B]), where [C][D] are product concentrations and [A][B] are reactant concentrations.
  • Under standard conditions, ΔG = ΔG°.
• ΔG is positive, -∆G depends on concentrations.
• If Keq > 1, products are favored at equilibrium.
• If Keq = 1, reactants and products are equally favored.
• If Keq < 1, reactants are favored at equilibrium.
• ΔG°': At standard conditions and pH 7.
  • Indicates the favored reaction direction (forward or reverse).
  • When ∆G=0, ∆G°'= - RTln [C][D]/[A][B] = -RTK'eq

Reaction Coupling

• Energetically favorable reaction is linked to an unfavorable (endergonic) reaction.
• Allows the overall reaction to proceed spontaneously.

Enzyme Kinetics Basics

• Active Site: Enzyme region where substrate molecules bind.
  • Highly specific to substrate types, creating a favorable environment.
  • Substrate binding leads to the transition state.
• Transition State: Molecular form that is neither substrate nor product.
  • It has the highest energy in the reaction pathway and is unstable.
• Enzymes facilitate transition state creation, and critical amino acids interact with substrate to accelerate product formation.
  • This releases binding energy, lowering activation energy.
• Binding Energy: Free energy released from enzyme-substrate interactions.
  • It's greatest at the transition state and lowers activation energy.
• Critical Residues: Crucial amino acids that interact with the substrate.
  • Making weak noncovalent interactions (hydrophobic, ionic, H-bonds) NOT covalent.
• Activation Energy (Ea): Necessary energy to form the transition state.
• Lock & Key Model: Enzyme and substrate fit precisely together.
• Induced Fit Model: Enzyme changes shape to accommodate the substrate.
• Enzyme-Substrate Complex: Enzyme + substrate.
• Enzyme Kinetics: Studies reaction rates in enzyme-catalyzed reactions.

Two Steps

• Formation of the enzyme-substrate complex. Product Formation.

Research & Experimentation

• Information learned from researching enzyme kinetics:
  • Enzyme chemical processes.
  • Enzyme function.
  • Biological relevance of enzymes.
• Experimental design for enzyme-catalyzed reactions:
  • What is being measured?
  • What is kept constant?
    • Temperature, pH, enzyme amount, time.
  • How are results displayed?
  • How are results interpreted?
  • How are calculations performed?
• Enzyme-Catalyzed Reaction: Pre-Steady State
  • Occurs very early in the reaction.
  • Enzyme-substrate starts to form.
• Steady State: Product formation at a constant rate and constant enzyme-substrate concentration.
  • Initial velocity is determined.
  • Equilibrium where substrate is reduced.
  • Less enzyme-substrate formation, and no significant changes in product/reactant formation.

Michaelis-Menten Kinetics

• Michaelis-Menten Assumptions: [S] > [E]
  • enzyme bound to the substrate is small.
  • only 1 substrate type present.
• Steady-State Assumption: [ES] is constant during the reaction, and ES formation/breakdown rates are equal.
• Initial Velocity, early in the experiment, [P] is very small.
  • This allows for the elimination of K2.
  • Binding step (E + S ↔ ES) is faster than the catalytic step (ES → E + P); the catalytic step is rate-determining.
• Km (Michaelis Constant): An enzyme/substrate constant.
  • Represents enzyme affinity for its substrate.
  • Measures needed substrate to reach ½ Vmax.
  • An intrinsic property unique to each enzyme.
  • Inversely proportional; higher Km means lower affinity.
  • Not affected by [E]. Unit: concentration units.
  • Substrate breakdown rate is represented by k-1 and k2
• Michaelis-Menten Equation: Describes how the initial reaction rate (V0) is influenced by the substrate concentration.
• Saturation Kinetics: Vmax plateaus, leading to zero-order reaction independent of [S].
  • In a first-order reaction, [S] < Km.
• kcat: Turnover number (Vmax/[E]), indicating enzyme's catalytic speed.
  • Measures substrate moles converted to product per second per active site.
• Catalytic Efficiency (kcat/Km): Accounts for enzyme-substrate interaction and catalysis efficiency.
  • A higher ratio means higher rate catalysis.
  • Velocity is proportional to [S].
• Hyperbolic Curve: Shows no cooperative binding and indicates one active site.

Line-Weaver-Burk Plot

• X-axis: 1/[S].
  • X-intercept: -1/Km
• Y-axis: 1/V.
  • Y-intercept: 1/Vmax
• Slope: Km/Vmax Note that catalytic efficiency is inverse.

Inhibition

• Irreversible Inhibitors: Bind covalently or noncovalently to the enzyme, leaving it nonfunctional even if dissociated.
• Reversible Inhibition: Rapid dissociation of the enzyme-inhibitor complex.

Types

• Competitive:
  • Inhibitor binds to active site, rendering the enzymes inactive and reducing available free [E] for binding.
  • Inhibitor presence in the area slows K1 and increases Km.
  • Sufficient substrate can outcompete the inhibitor and vmax is unchanged.
• Noncompetitive:
  • Inhibitor interacts with the enzyme allosteric site OR the ES complex.
  • Active site is not directly affected by the inhibitor, but the enzyme conformation is changed by it reducing enzymes available for catalysis.
  • E, S, and ES are removed from the concentration at the same ratio.
  • A noncompetitive inhibitor cannot be overcome by increasing [S].
• Uncompetitive:
  • Inhibitor binds to the ES complex allosteric site, distorting the enzyme active site and rendering the enzyme ineffective.
  • Decreases [ES], causing decreases to both Km and vmax as more E and S react and increase Ki.
  • An uncompetitive inhibitor cannot be reversed by increasing [S].

Inhibition Type Effects

• Competitive: Km (apparent) increases and Vmax (apparent) shows no change.
  • Adding sufficient substrate will eventually allow the reaction to reach Vmax
• Noncompetitive: Km (apparent) shows no change, and Vmax (apparent) decreases.
  • It has less active sites functional enough to make products.
• Uncompetitive: Km (apparent) decreases, and Vmax (apparent) decreases.
  • Has les functional active sites to make products.

Allosteric Enzymes

• Have multiple active sites.
• Sigmoidal Curve: Cooperative binding for its multiple active sites and subunits.
• Activity is controlled by allosteric effectors (inhibitors or activators).
• Feedback Inhibition: Products influence reactant release.
• Affects Km, Vmax, or both.

Catalytic Strategies

• Covalent Catalysis: Active site contains a nucleophile covalently modified.
• General Acid-Base Catalysis: Molecule aside from water donates or accepts a proton.
• Catalysis by Approximation: Enzyme brings two substrates together for catalysis.
• Metal Ion Catalysis: Metal ions aid catalysis, e.g., as an electrophilic catalyst.

Lysozyme Background

• Cleaves glycosidic bonds in bacterial cell walls.
  • Can cleave at multiple points.
• Substrate: Bacterial cell-wall polysaccharide chains consisting of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).
• Scissile Bond: A chemical bond that is broken by the enzyme.

Lysozyme Structure

• Mostly alpha helices with some beta sheets.
• Active Site: Six-sugar subunits fit – labeled A-B-C-D-E-F.

Reaction Summary

• The bond between D and E is broken.
• Critical amino acids: Asp and Glu.
• Substrate is stabilized by non-covalent interactions (H-bonds) and van der Waals forces. Ex, two tryptophans and aspartic acid.
• Creates A-B-C-D and E-F products, where ONLY 1 BOND is BROKEN.
  • Red: Lysozyme amino acids in the active site.
  • Blue: Substrate atoms for H-bonds.
• Class Scenario: For R-groups that can make H-bonds, substrate binding has to occur in the amino acids in the area where R groups are bound in that location.
  • If these amino acids are replaced with nonpolar , no binding will occur.
• Numbering: Number next to amino acids shows its sequence position. Ex, Asp 101 = Aspartic acid in the one hundred an first position.
• Folding allows close proximity of various number amin acids.

Lysozyme Mechanism

• General acid-bas catalyzed.
• Substrate sits in the active site, but lysozyme distorts the sugar in the D site allowing to cut the sugar chain in D and E.
• Step 1: Glu 35 acts as acid and donates protons from the r-group to the substrate, then breaks the glycosidic bond b/w A-B-C-D and E-F
  • Product with E-F leaves attracting proton charge to the electron of Glu.
• Step 2: Negative Asp 52 charge stabilizes Carbonium D's A-B-C-D
• Step 3: Glu 35 acts as base and deprotonates the water.
  • Hydroxide attacks carbonium for substrate release.
  • Amino Acids now reset in the new active set.

Chymotrypsin Background

• Protease: Enzymes that breakdown peptide bonds.
  • They need specific molecular features to cleave bonds.
• Serine Protease: Enzymes that use serine to breakdown peptide bonds.
  • Chymotrypsin is secreted in the pancreas and acts in the small intestine.
  • Released as a zymogen
• Zymogen: proteins inactive form, must cleave.
• Hydrolyzes a peptide bond on the C-terminus of large hydrophobic residues of phenylalanine, tryptophan, tyrosine, and methionine.
• Chymotrypsin finds the specificity pocket by looking for said pocket
• • Covalent Catalysis

General Covalent Catalysis

• Step 1: acylation -> acyl enzyme intermediate formation
• Step 2: deacylation -> free enzymes.

Chymotrypsin Active Site Overview

• Composition: three chains , two domains, and three key residues.
• Specificity pocket
• Catalytic Triad
• Oxyanion Hole: Main chain assists in peptide bonds
  • Glycine present in the structure
• Class scenario: if a glycine R group is in the enzyme cavity for bonding, the corresponding acids will break

Catalytic Triad Info

• Amino Acids responsible for destroying Amino Peptide bonds in active residue
  • Residues Key are Aspartic acid, histidine, serine with 2 domains
• Key residues Serine, histadine, aspartic acid.
• Active site with no substrate -> Hydrogen bonds come from amino acids to sustain Triad.
• Active set with substrate
  • HIs acts as Catalytic bas
  • Ser 195's hydroxyl -> more Alkoxide reactive

Chymotrypsin Mechanism

• Covalent Catalysis.

• Phase 1: Creation is Enzymes -Substrate Intermediate.

  • Enzymes Substrate Bonding, a huge hydrophobic substrate sit in specificity. • Chymotrypsin can locate the pockets of the film

• 102 APS function well, setting and fixing the residue of histidine.

• Step 1 HO: HIs57 deprotonated Ser195 creating Alkoixe results with highly reactive Alkoxide for nu attack with carbine carbon from the substrate

• Step 2 carbonyl -> Kicks of amino as a part group

  • Acyl -> connected to 195 in set.

• Phase 2 2 acylation - free enzymes.

• Step 3 the incoming H20 molecules use a - from Hi

  • L- hydroxyl carbon attack Acyl

• Carbonly double bonds stabilize hydrogen

• I formed tetraledral intermediate. ->

• Carbonyl recreates Ser in kick forms Triad products.

• Is carboxyl for serine release, reseted Triad ->

Carbonic Anhydrase Background

• Found in RBC, mucosa, pancreas and renal tubules.
• Carbonic Acid maintains the Blood level _ site-> metal cofactor: Zinc, lowers PKA to assist water dis-association-> negative OH groups, helps assist with binding.
• The close location to zinc binding helps assist binding locations to carbon (close prox) -> and helps in assistance in active sights and residues of Histine.

Carbonic Anhydrase Mechanics

• Step 3: Metal Ion catalyst helps in approximation in catalytic.
• Rate the reaction of limit steps.

Enzymes-Activity depend on Temperature and pH

• High temps-> faster -> disrupt temp and bonding and influence interactions, leading to conformation.
• Cold Temps-> causes less interactions and therefore less probability of subs binding.
• pH value changes behavior and group binding to terminals - and tert bonds.
• the wrong form causes Active site repulsion and incorrect interaction with enzymes.

Regulatory Strategies

• Allosteric
• Many different forms.
• Revers covalent mod and Proto activated.
• enzymes controll -> express

Aspartate Transcarbamolase

• Pyrimidine synthesis-> essential
• Give blocks Catalyse-> a non energy step
• used to nuclear product Do mechanics. Composed for 6 structures Organ-> tri meters the middle 6 of them 3 structures -> Active sub units.

Regulation

• zinc _ sites. Act ATP regulators T state

Enzymes- Activity depend on Temperature and pH

• High temps-> faster -> disrupt temp and bonding and influence interactions, leading to conformation. Cold Temps-> causes less interactions and therefore less probability of subs binding. pH value changes behavior and group binding to terminals - and tert bonds.
• the wrong form causes Active site repulsion and incorrect interaction with enzymes.

Regulatory Strategies

• Allosteric
• Many different forms
• Revers covalent mod and Proto activated.
• enzymes controll -> express

Aspartate Transcarbamylase

• pyrimidine synthesis-> essential

• catalyzing

• Do regulations Composed for 6 structures Organ-> tri meters the middle 6 of them 3 structures -> Active sub units

Enzyme Regulations

• Zn Sites act ATP regulators

• T state

Protein Kinase A –

• Covalent Mod

  • phosphorylates

• not reversible

• High energy _ subunits. High energy

  • High stress