Enzyme Notes

Enzymes: Basic Concepts

Intended Learning Outcomes

• Understand the basic concepts of what an enzyme is.
• Understand what an enzyme does.
• Understand the optimum conditions for enzymes to work.
• Understand the concept of free energy.
• Understand methods of reactant binding.
• Understand strategies enzymes can use, with examples like chymotrypsin and carbonic anhydrase.

What are Enzymes?

• Enzymes are specialized proteins.
• They recognize a specific chemical structure in the presence of similar structures to produce a specific product.
• Enzymes are efficient biological catalysts.
• Catalysts increase the rate of a chemical reaction without being changed at the end of the reaction.
• Almost all enzymes are proteins.
• A few enzymes also contain RNA and are called ribozymes (e.g., the ribosome).

Enzymes Speed Up Reaction Rates

• Enzymes can significantly increase the rate of a reaction.
• Example: A reaction occurs 10,000,000 (10^7) times faster with an enzyme.
• Enzymes can increase the rate of a reaction by a million times or more.
• Carbonic anhydrase, present in blood, catalyzes the hydration of CO_2.

Enzyme Classification

• Systematic names for enzymes include the substrate and the type of reactions, and the enzyme name often ends in ‘ase’ (e.g., ATP synthase – synthesizes ATP).
• Oxidoreductase: Catalyzes oxidation-reduction reactions.
• Transferase: Transfers chemical groups from one molecule to another, involving two substrates and producing two products.
• Kinase: Adds a phosphate group.
• Phosphatase: Removes a phosphate group.
• Lyases: Catalyze a C-C bond or C-N cleavage or release CO_2 from a β-keto acid.
  • Important neurochemical reactions, including the formation of dopamine and serotonin, are examples of this reaction.
• Synthase: Used if an NTP is not involved in new bond formation.
• Synthetase: Used if ATP is required.
  • In practice, these terms are often used interchangeably.
• Isomerase: Changes molecules into different isomers (move chemical groups around).
• Ligase: Ligates (joins) two molecules.

Factors Affecting the Rate of an Enzyme-Catalyzed Reaction: Temperature

• Most reactions occur faster at higher temperatures.
• At higher temperatures, molecules have more energy, making it easier to overcome the activation energy barrier.
• Above a certain temperature, the protein will denature (unfold) and become inactive.

Enzyme Activity and Temperature

• Enzyme activity typically increases with temperature until it reaches an optimum point.
• Beyond this point, the enzyme starts to denature due to the heat, leading to a rapid decrease in activity.

pH

• Most enzymes have an optimum pH.
• Ionizable amino acids in the protein must be in the right state for the enzyme to work.
• Optimum pH is suited to their environment.
  • In the stomach, the pH is around 1-2.
  • In the upper intestine, the pH is around 8.

Effect of pH on Carbonic Anhydrase Activity

• Carbonic anhydrase activity is maximally active at high pH.
• The reaction catalyzed by carbonic anhydrase:
  CO2 + H2O \rightleftharpoons H2CO3 \rightleftharpoons HCO_3^- + H^+

Effect of pH

• Extremes of pH generally inactivate enzymes.
• pH optimum: Maximum activities are typically between pH 4.5 – 8.0.
• Enzymes have a narrow pH range.
• Exceptions:
  • Pepsin: pH 1.8
  • Trypsin: pH 9.8

Enzymes are Highly Specific

• Enzymes have a high degree of specificity.
• The chemical reaction catalyzed by a certain enzyme always involves the same starting chemical (the substrate) and produces the same product.
• This precise specificity is defined by the 3-dimensional structure of the enzyme protein.

Proteases and Specificity

• Proteases catalyze proteolysis (lysis – breaking, proteo - proteins).
• They break peptide bonds by hydrolysis (breaking with water).

Different Protease Enzymes Have Different Specificity

• Trypsin: Cleaves only after arginine or lysine residues.
• Thrombin: Cleaves between arginine and glycine only in particular sequences.
• Chymotrypsin: Cleaves on the COOH side of bulky aromatic and hydrophobic amino acid residues.
• Papain: Cleaves all peptide bonds irrespective of sequence.

Enzymes and Free Energy

• Enzymes can only speed up the rate of a chemical reaction.
• They cannot change whether or not the reaction will occur spontaneously.
• Whether a reaction occurs spontaneously is determined by the free energy change of the reaction, \Delta G.
• The study of free energy of reactions is called thermodynamics.

Thermodynamics

• Energy is required to form both the starting material of a reaction and the product of a reaction.
• The total free energy of a molecule (G) cannot be measured directly.
• The change in free energy (\Delta G) of a reaction is the difference in G between the reactants and products.
• \Delta G CAN be measured.

Free Energy Change

• If \Delta G is negative:
  • Reaction can occur spontaneously (without energy input).
  • This is called an exergonic reaction.
• If \Delta G is positive:
  • Reaction cannot occur unless energy is provided.
  • This is called an endergonic reaction.
• If \Delta G = 0:
  • Reaction is at equilibrium.
  • There is no net change in the amount of products or reactants.

Exergonic Reactions

• Exergonic reactions release energy, resulting in a negative \Delta G.
• The products have lower free energy than the reactants.

Endergonic Reactions

• Endergonic reactions require energy input, resulting in a positive \Delta G.
• The products have higher free energy than the reactants.

\Delta G is Independent of Reaction Path (Mechanism of Reaction)

• The free energy change between reactants and products remains the same regardless of the reaction pathway.

\Delta G and Reaction Rates

• A negative \Delta G indicates that a reaction can occur spontaneously without extra energy input.
• \Delta G tells us nothing about the rate of reaction.
• It might take 10,000 years to reach equilibrium (for the reaction to finish)!

Calculating Free Energy

• Cannot measure absolute free energy (G) for a chemical species.
• But, we CAN measure the change in free energy (\Delta G) for a chemical reaction.

Equilibrium Constant

• A reaction is in equilibrium when there is no net change in the concentrations of reactants and products, and the reaction has finished.
• Example reaction: A + B \rightleftharpoons C + D

Equilibrium Constant Definition

• The ratio of concentrations of products and reactants at equilibrium is constant.
• Defined as the equilibrium constant, K_{eq}.
• K_{eq} = \frac{[C][D]}{[A][B]}
• For the reaction: A + B \rightleftharpoons C + D

Calculating Free Energy

• For the reaction: A + B \rightleftharpoons C + D
• The free energy change is given by: \Delta G = \Delta G^o + RT \ln \frac{[C][D]}{[A][B]}
  • Where \Delta G^o is the standard free energy.
  • R is the gas constant, 8.315 \text{ J.mol}^{-1}.
  • T is the temperature in kelvins (K), 25 ^\circ C = 298 \text{ K}.

Constants and Units

• \ln means the ‘natural log’ = \log_e
• [C] means the concentration of chemical C, in M (molar) or mol.l-1
• R is the gas constant, 8.315 \text{ J.mol}^{-1} \text{K}^{-1}
• A Joule (J) is a measure of energy
• 1kJ (kilojoule) = 1000 J

Standard Free Energy

• Standard free energy, \Delta G^o is the free energy change under conditions where all components started at 1M (1 mol/l).
• A more useful term is \Delta G^{o'} (“delta G nought prime”) – the standard free energy change at pH 7.
• All components started at 1M except H+ which started at 10^{-7} M.
• For the reaction: A + B \rightleftharpoons C + D

Free Energy at Equilibrium

\Delta G = \Delta G^{o'} + RT \ln \frac{[C][D]}{[A][B]}
At equilibrium, \Delta G = 0
0 = \Delta G^{o'} + RT \ln \frac{[C][D]}{[A][B]}
\Delta G^{o'} = -RT \ln \frac{[C][D]}{[A][B]}

Standard Free Energy and Equilibrium Constant

\Delta G^{o'} = -RT \ln \frac{[C][D]}{[A][B]}
K{eq} = \frac{[C][D]}{[A][B]} \Delta G^{o'} = -RT \ln K'{eq}

Calculating \Delta G^{o'}

\Delta G^{o'} = -RT \ln K'_{eq}
So, if we can measure the concentrations of reactants and products at equilibrium, we can calculate \Delta G^{o'}.