Enzymes, Catalysts, and Thermodynamics: Biochemistry Fundamentals

What is a catalyst?

A substance that speeds up a reaction and does not get used up in the reaction.

What are enzymes?

Proteins that fold into catalytically active shapes and act as biological catalysts.

Are all enzymes proteins?

No — all enzymes are proteins, but not all proteins are enzymes.

How do enzymes catalyze reactions?

Through a diverse set of mechanisms, acting like small molecular machines.

What is a substrate?

The molecule that an enzyme typically acts on in a biological context.

What is a product?

The molecule produced after the enzyme catalyzes the reaction.

What is an active site?

A substrate "pocket" in the enzyme's tertiary or quaternary structure where substrates bind.

What happens if you remove the metal cofactors from an enzyme that needs them?

The enzyme will no longer function properly.

In the bacterial enzyme organophosphate hydrolase (OPH), what do the amino acids in the active site do?

They coordinate two zinc atoms to hold them in position to participate in catalysis.

What are cofactors?

Non-protein molecules or ions that help enzymes catalyze reactions.

What are the three main types ("flavors") of cofactors?

1) Metal ions (e.g., Zn²⁺, Fe²⁺), 2) Cosubstrates (e.g., NAD⁺, Biotin), 3) Prosthetic groups (e.g., Heme).

What are metal ion cofactors?

Metal ions such as Zn²⁺, Mg²⁺, Ni²⁺, Mo⁶⁺, or Mn²⁺ that assist in catalysis by stabilizing structures or participating in reactions.

What are coenzymes?

Organic cofactors derived from vitamins that assist enzymes in catalysis.

What is the difference between cosubstrates and prosthetic groups?

Cosubstrates (e.g., NAD⁺, Biotin) bind temporarily, while prosthetic groups (e.g., Heme) are strongly bound to the enzyme.

What is an apoenzyme?

The inactive protein portion of an enzyme that requires a cofactor to become active.

What is a holoenzyme?

The active form of the enzyme containing both the protein and its cofactors.

What happens if a cofactor is removed from an enzyme?

The enzyme becomes inactive (apoenzyme form).

What is the function of rare metals like Mo and Se in enzymes?

They serve as essential cofactors in specialized enzymes such as nitrogenase and glutathione peroxidase.

What is the main function of enzymes?

To enhance (speed up) the rate of biochemical reactions.

Does the presence of an enzyme make a reaction occur that wouldn't happen otherwise?

No — the reaction would still happen, but much more slowly without the enzyme.

How do enzymes affect reaction rates?

They increase the reaction rate by lowering the activation energy barrier.

What is meant by "rate enhancement"?

The ratio of the catalyzed rate (kcat) to the uncatalyzed rate (kun), showing how much faster the enzyme makes the reaction.

What does carbonic anhydrase catalyze?

The reversible conversion of CO₂ + H₂O ↔ H₂CO₃ ↔ H⁺ + HCO₃⁻.

What is the relationship between enzyme presence and reaction speed?

With an enzyme → fast reaction; without enzyme → slow but still occurs.

What is the equation for Gibbs Free Energy?

ΔG = ΔH - TΔS

What does ΔH represent?

Change in enthalpy — the amount of energy or heat taken up by bond formation.

What does ΔS represent?

Change in entropy — a measure of disorder or randomness in the system.

What does T represent in the Gibbs equation?

Temperature, which acts as a multiplier on entropy.

What makes a reaction more favorable according to Gibbs Free Energy?

A lower (more negative) ΔG value.

What does a negative ΔG indicate?

The reaction is spontaneous and thermodynamically favorable.

What is the formal definition of Gibbs energy?

The thermodynamic potential that is minimized when a system reaches chemical equilibrium at constant pressure and temperature.

What happens when ΔG is more negative?

The reaction produces more product at equilibrium and is more favorable.

What does ΔG° mean?

Change in Gibbs Free Energy under standard conditions.

What does ΔG°′ mean?

Change in Gibbs Free Energy under standard cellular conditions (pH ≈ 7, constant [H₂O]).

Do enzymes change the ΔG of a reaction?

No, enzymes do not change ΔG; they only change the rate at which equilibrium is reached.

Is the product concentration at equilibrium different for catalyzed vs. uncatalyzed reactions?

No, it is the same at equilibrium — enzymes only affect rate, not equilibrium position.

What are the three factors ΔG depends on?

ΔH (enthalpy), ΔS (entropy), and T (temperature).

What is the equation that relates Gibbs Free Energy to the equilibrium constant?

ΔG°′ = -RT ln K′eq

What happens to ΔG°′ when K′eq is large (≫1)? N(Gibbs equation)

ΔG°′ becomes very negative, meaning the reaction strongly favors products.

What happens to ΔG°′ when K′eq is small (≪1)? (Gibbs equation)

ΔG°′ becomes positive, meaning the reaction favors reactants.

What is the relationship between ΔG°′ and K′eq?

The relationship is logarithmic and nearly linear across magnitudes of K′eq.

How much does ΔG°′ change for every 10-fold (1 order of magnitude) change in K′eq?

ΔG°′ changes by approximately 5.69 kJ/mol.

What does it mean when K′eq = 1?

ΔG°′ = 0, indicating equilibrium (equal concentrations of products and reactants).

What does a negative ΔG°′ indicate about equilibrium?

Products are favored at equilibrium.

What does a positive ΔG°′ indicate about equilibrium?

Reactants are favored at equilibrium.

What does ln K′eq represent in the equation?

The natural logarithm of the equilibrium constant, showing how far the equilibrium lies toward products or reactants.

What happens to ΔG°′ if the concentrations of products are high compared to reactants?

ΔG°′ becomes more negative, favoring product formation.

What does the activation energy (Ea) represent?

The energy barrier that must be overcome for a reaction to occur.

How do enzymes affect the activation energy?

Enzymes lower the activation energy (Ea) by reducing the height of the energy barrier ("the hill").

What does ΔG‡ represent?

The free energy of activation — the difference in energy between reactants and the transition state.

How do enzymes reduce ΔG‡?

By stabilizing the transition state, making it easier for reactants to form products.

Do enzymes change the ΔG (overall free energy change) of a reaction?

No, enzymes do not change ΔG — they only affect the rate of the reaction.

What is the result of lowering the activation energy?

The reaction proceeds faster because more reactant molecules can reach the transition state.

How do enzymes stabilize the transition state?

By forming weak interactions with it that lower its energy and make it easier to achieve.

Does lowering activation energy affect the equilibrium position?

No, equilibrium remains the same; both forward and reverse reactions are accelerated equally.

What does the "diamonds are forever" example illustrate?

Diamonds are thermodynamically unstable and will eventually convert to graphite, but the process is extremely slow due to a high activation energy.

Why don't we observe diamond-to-graphite conversion in our lifetime?

The reaction's activation energy is so high that the rate is effectively negligible over human timescales.

What is the effect of an enzyme on the "hill" in the reaction energy diagram?

It reduces the size of the hill (the energy barrier), allowing the reaction to occur more readily.

What happens if a reaction has a negative ΔG but a high activation energy?

The reaction is spontaneous but occurs very slowly without a catalyst.

How can enzymes help in spontaneous reactions with high activation energy?

Enzymes lower the activation energy, allowing the reaction to occur faster.

What determines if a reaction is spontaneous or not?

The sign of ΔG — negative means spontaneous, positive means nonspontaneous.

Why might a spontaneous reaction take a long time to complete?

Because it may have a large activation energy barrier that slows down the rate.

Do enzymes change the position of equilibrium?

No — enzymes do not change the equilibrium concentrations of reactants or products.

What does V₀ represent?

The initial velocity (rate) of the reaction.

How does the presence of an enzyme affect V₀?

It increases V₀, meaning the reaction reaches equilibrium faster.

What happens to V₀ as substrate concentration [S] increases?

V₀ increases proportionally at first — the initial slope becomes steeper.

What does the graph comparing "No enzyme" vs "+ Enzyme" show?

Both reach the same equilibrium product concentration, but the enzyme-catalyzed reaction does so much faster.

Does an enzyme affect both forward and reverse reactions?

Yes — enzymes speed up both directions equally, maintaining the same equilibrium ratio.

What does it mean that enzymes "facilitate equilibrium"?

They simply help the system reach equilibrium faster without altering the final balance of reactants and products.

What happens if more substrate is added?

The equilibrium concentration of product increases and the initial velocity (V₀) becomes faster.

What determines the slope of the reaction progress curve?

The initial substrate concentration [S] — higher [S] produces a steeper initial slope (higher V₀).

What is the key takeaway about enzymes and equilibrium?

Enzymes accelerate the rate of approach to equilibrium but do not change the equilibrium position itself.

What happens to equilibrium concentration when substrate concentration increases?

The equilibrium is reached at a higher product concentration because more substrate is available to convert.

Why does the line eventually flatten in both cases (with and without enzyme)?

Because the reaction reaches equilibrium — the rates of the forward and reverse reactions become equal.

How is V₀ related to substrate concentration [S]?

Initially, V₀ ∝ [S] — as substrate concentration increases, the reaction starts faster.

Why does the reaction rate slow down as it approaches equilibrium?

Because substrate is being consumed and product accumulation leads to the reverse reaction balancing the forward one.

What does kcat represent?

The turnover number — how many substrate molecules one enzyme molecule converts to product per second.

What does Vmax represent?

The maximal velocity of the reaction when all enzyme active sites are saturated with substrate.

What is the relationship between kcat and Vmax?

kcat = Vmax / [E]ₜ

What does the shape of the reaction velocity vs. substrate concentration curve show?

The reaction rate increases with [S] until it levels off at Vmax when the enzyme becomes saturated.

Why does the curve plateau at Vmax?

Because all enzyme active sites are occupied — adding more substrate won't increase the rate.

What is the meaning of the enzyme-substrate (ES) complex?

It represents the intermediate formed when enzyme binds substrate before product formation.

Why is kcat considered a constant?

Because it normalizes Vmax for enzyme concentration — it reflects the intrinsic catalytic speed of each enzyme molecule.

What determines how "fast" an enzyme is?

Its kcat value — a higher kcat means the enzyme converts substrate to product more quickly.

What happens to Vmax if enzyme concentration doubles?

Vmax also doubles, since it depends on the number of available enzyme active sites.

What happens to initial velocity (V₀) as substrate concentration [S] increases?

V₀ increases and eventually converges to a maximum value (Vmax).

What does the left-hand graph (product vs. time) illustrate?

Increasing [S] causes reactions to start faster (higher slopes), but they all level off once equilibrium is reached.

What does the right-hand graph (V₀ vs. [S]) illustrate?

Reaction velocity rises with substrate concentration and plateaus when the enzyme becomes saturated — reaching Vmax.

What does it mean for an enzyme to be "saturated"?

All enzyme active sites are occupied by substrate; adding more substrate cannot increase the rate.

Why can't an enzyme speed up the reaction beyond Vmax?

Because every enzyme active site is already working at full capacity.

How is Vmax related to enzyme concentration?

Vmax is proportional to total enzyme concentration — doubling enzyme doubles Vmax.

Is Vmax a true constant?

No, it depends on enzyme concentration. Only kcat is a true constant intrinsic to the enzyme.

How is kcat calculated?

kcat = Vmax / [E]ₜ

What does the idea of "slopes converging" in the left graph mean?

As [S] increases, initial velocities (V₀) approach a limiting value, Vmax.

What happens if the amount of enzyme in an experiment is doubled?

All reaction rates double, resulting in a new, higher Vmax.

How is Vmax determined experimentally?

By measuring reaction velocities (V₀) at increasing [S] until the curve levels off.

When should kcat be used instead of Vmax?

When comparing enzyme efficiency independently of enzyme concentration.

What does the plateau on the V₀ vs. [S] graph represent?

The enzyme is saturated — reaction velocity has reached its maximum (Vmax).

What does turnover number represent?

The number of substrate molecules converted to product per enzyme molecule per second under saturating substrate conditions.

What is another term for turnover number?

kcat.