Free Energy, Enzymes, and Reactions

• Chapter 8 discusses the relationships between free energy, activation energy, transition state, and the role of enzymes.

Free Energy

• It's the measure of mechanical or chemical work extracted from a system or reaction.
• Spontaneous processes have a negative change in free energy, releasing energy (exergonic).
• Gibbs Free Energy must be negative for a reaction to occur and proceed forward.
• Entropy increases in spontaneous (exergonic) reactions and decreases in nonspontaneous (endergonic) ones.
• Gibbs Free Energy is negative for spontaneous reactions, positive for endergonic, and zero at equilibrium.
• Phosphodiester bond formation is nonspontaneous but becomes spontaneous when coupled with a hydrolysis reaction.

Enzymes and Reactions

• Enzymes increase reaction rates of already possible reactions, without forcing them to occur.
• They lower activation energy by stabilizing the transition state.
• The activation energy amount doesn't affect Gibbs Free Energy; it influences the energy difference between products and reactants.
• Enzymes can accelerate reactions but cannot alter the Gibb's free energy.
• No amount of enzyme can force an unfavorable reaction to occur.

Activation Energy

• Activation energy starts the reaction; enzymes reduce this energy for quicker reactions.
• Enzymes reduce activation energy by stabilizing the transition state.
• The energy to reach the transition state is called the Gibbs Free Energy of Activation.
• Enzymes reduce the Gibbs Free Energy of Activation but not the overall Gibbs Free Energy.
• Enzymes alter reaction rates, not outcomes, following thermodynamics.
• Positive Gibbs Free Energy reactions won't proceed without specific conditions: substrate and products not in equilibrium, or coupling with favorable reactions.
• Coupling endergonic reactions with exergonic ones makes the overall Gibbs Free Energy negative.
• Enzymes facilitate equilibrium by aligning substrates, functional groups, desolvation, and inducing strain.

Impact of Active Sites

• The enzyme stabilizes the transition state by forming the enzyme-substrate complex.
• The energy for unfavorable processes comes from the interaction of amino acids in the active site.
• Active sites feature covalent catalysis and noncovalent interactions from the enzyme and substrate's amino acid sequence.
• The negative Gibbs Free Energy arises from reversible non-covalent interactions between the enzyme and substrate.
• Enzymes aid transition state formation through numerous weak, noncovalent interactions.
• Enzymes accelerate reactions by lowering activation energy, but they cannot force an energonic reaction to happen, just speed up exergonic reactions.
• Enzymes change reaction rates without changing equilibrium, therefore not defying thermodynamics.
• Under physiological conditions, substrate and product concentrations aren't at equilibrium, affecting Gibbs Free Energy.
• Gibbs Free Energy = Gibbs Free Energy of activation + RTln (Keq).
• Enzymes decrease reaction time and accelerate reactions by stabilizing transition states.
• Therefore, decreasing Gibbs Free Energy of activation.

Free Energy Diagrams

• Exergonic reactions: Products lower in energy; K'eq > 1; negative Gibbs Free Energy of activation; favored forward reaction; entropy increases.
• Endergonic reactions: Products higher in energy; K'eq < 1; positive Gibbs Free Energy of reaction; favored reverse reaction; entropy decreases; energy required.

Enzyme-Substrate Interaction Models

• Emil Fischer's Lock and Key model (1890): Exact arrangements of substrate in the active site.
• Dan Koshland's Induced Fit model (1958): Substrate binding causes enzyme shape change, reinforcing noncovalent interactions.

Enzyme Kinetics

• Many enzymes follow Michaelis-Menten kinetics; the reaction rate increases with substrate until saturation.
• Low substrate = slow reaction; high substrate = fast reaction, up to enzyme saturation.
• V0 (initial reaction velocity) is fast initially but slows as substrate decreases and product increases.
• VO is the observed initial linear velocity when [P] = 0.
• Km (Michaelis constant) = (k2 + k-1) / k1.

Km and Vmax

• K1 combines enzyme and substrate; K2 forms enzyme and product; K-1 reverses the enzyme-substrate complex.
• Km determines reaction evaluation without additional variables.
• Vmax (maximum velocity) is the reaction's limit, rarely reached due to substrate binding and product formation already happening.
• Vmax is measured in mM/sec; Km is substrate concentration at half Vmax (Km = Vmax/2).
• Vmax requires plot; Km value indicates particular enzyme and substrate characteristics.
• Km is [S] at Vmax/2 in mM; Vmax is reaction rate in mM/sec.
• Small Km: enzyme needs little substrate for maximum velocity, so a high binding affinity.
• Large Km: enzyme needs much substrate for maximum velocity, so lower binding affinity.
• Use this to choose how much Substrate to use.

Lineweaver-Burk Plots

• Lineweaver-Burk plot: double reciprocal plot; y-intercept as 1/Vmax, x-intercept as -1/Km.
• Vmax determined via y-intercept and Km is calculated from slope.
• Smaller Km means stronger binding affinity, and higher Km means weaker affinity.
• Km is [S] at 1/2 Vmax or Vmax/2.

Kcat and Kinetic Perfection

• Kcat are the measurements.
• Kcat (turnover number): Substrate molecules one enzyme molecule converts to product per second when saturated.
• Kcat measures catalytic activity by low Km (high binding affinity); high Km suggests needing much substrate (low binding affinity).
• Bigger ratio Kcat/Km performs the better under physiological control.
• Kinetic perfection occurs when the reaction rate is limited by only substrate diffusion.
• Enzymes with complicated mechanisms have similar constants: Vmax = kcat [Etotal], and the ratio is still kcat/Km.

Enzyme Inhibition and Activation

• Lineweaver-Burk plots assesses the mechanisms of enzymes to find inhibition and activation.
• Allosteric enzymes don't follow Michaelis-Menten kinetics, use s-shaped sigmoidal curves from activators and inhibitors.
• Allosteric enzymes have multiple subunits and are regulated.

Reversible and Irreversible Inhibition

• Allosteric enzymes have more than one subunits and multiple binding sites.
• Substrate binding boosts the affinity of the second substrate and shows cooperativity (sigmoidal curve).
• With binding of regulatory molecules (activators and inhibitors) they can influence the active substrate by shifting left or right on the active site.
• Noncompetitive inhibitors have a different binding site on the active site.
• The equilibrium shifts when regulatory enzymes bind the active site; decreasing (inhibitors) or increasing (activators) binding reactions.
• Competetive inhibitors and substrates compete for the active site.

More on regulatory sites

• If there is no inhibitor the substrate with bind directly to the active site.
• Reversible inhibitors a substance can bind to inhibit, but the enzyme can still be released, and unbound to the active site.
• Allosteric enzymes binds at the regulatory sites, if the concentration is increased, then substrate concentration must increase increase.
• Enzymes accelerate reactions without changing equilibrium.
• Physologic [S] and [P] are not in perfect equilibrium which affect the Gibbs free energy of activation for reaction.

Enzyme Inhibition

• Enzyme inhibition does 2 things: decrease enzymes ability to bind to substrate or decrease enzyme catalyic activity or turnover.
• Most drugs inhibit enzymes, scientists test drugs to evaluate effectiveness.
• There are two type of inhibitors: reversible and irreversible
• Reversible competitors can be non-competitve or competitive.

Lineweaver-Burk Plot

• Km gets lowered , Vmax gets decreases when it can't be carried out.
• Allosteric inhitors are not static.
• suicide molecules inhibit the molecules and may resemble S group, with some of the proteins.

Know 4 Catalytic Stategies

• Covalent Catalysis
• General Acid-Base Catalysis
• Metal Ion Catalysis -Catalysis with approimation.

Activation of water

• Water is very important in many chymotrypsin reactions to activate what is needed.
• Cysteine Proteases and aspatrtly proteases are catalytic diads.
• Cysteine Proteases needs cystine and hdristine.
• Active sites can only have acid base or water molecules to activate these.