Biochemistry Lecture 14

Enzymes are proteins

Enzymes are mostly proteins that catalyze biological reactions

Catalysis definition

Enzymes speed up reactions without being consumed

Biological reactions

Reactions under biological conditions are inherently slow

Effect of enzymes on rate

Enzymes increase reaction rates by many orders of magnitude

Reactant definition

Starting molecule in a reaction

Product definition

Molecule formed from a reaction

Substrate definition

The reactant molecule that binds to an enzyme

Basic enzyme reaction

Substrate + Enzyme ⇌ Product + Enzyme

Enzyme reuse

Enzymes are not consumed and can be reused after reaction

Enzyme classification basis

Enzymes are classified by the type of reaction they catalyze

Oxidoreductases

Transfer electrons or hydrogen atoms

Transferases

Transfer functional groups between molecules

Hydrolases

Catalyze hydrolysis reactions involving water

Lyases

Cleave bonds or add groups to double bonds without hydrolysis

Isomerases

Rearrange atoms within molecules

Ligases

Join molecules using ATP energy

Translocases

Move molecules or ions across membranes

EC number definition

Numerical system used to classify enzyme function

Generic enzyme names

Names like trypsin do not describe function

Cofactor definition

Non-protein molecule required for enzyme activity

Holoenzyme

Active enzyme including cofactor and apoenzyme

Apoenzyme

Protein portion of enzyme alone

Metal ion cofactors

Inorganic ions assisting catalysis

Examples of metal ions

Cu²⁺, Fe²⁺/Fe³⁺, Mg²⁺, Mn²⁺, Zn²⁺, Ni²⁺, Mo, K⁺

Prosthetic group

Cofactor tightly or permanently bound

Coenzyme

Organic cofactor that binds reversibly

Biocytin function

Transfers CO₂

Coenzyme A function

Transfers acyl groups

Coenzyme B12 function

Transfers hydrogen atoms and alkyl groups

FAD function

Transfers electrons

Lipoate function

Transfers electrons and acyl groups

NAD⁺ function

Transfers hydride ions

Pyridoxal phosphate function

Transfers amino groups

Tetrahydrofolate function

Transfers one-carbon units

Thiamine pyrophosphate function

Transfers aldehyde groups

Transition state definition

Unstable intermediate between substrate and product

Reaction pathway

S ⇌ TS ⇌ P

Activation energy (ΔG‡)

Energy required to reach transition state

Biochemical standard conditions

298 K, 101 kPa, pH 7

Exergonic reaction

Negative ΔG, releases energy

Endergonic reaction

Positive ΔG, requires energy

Energy relationship

Substrate has higher free energy than product in exergonic reactions

Thermodynamics definition

Study of spontaneity and equilibrium

Kinetics definition

Study of reaction rates

Thermodynamics predicts

Spontaneity and equilibrium position

Thermodynamics limitation

Does not predict rate or mechanism

Equilibrium constant (Keq)

Ratio of products to reactants

ΔG relationship

ΔG°′ = −RT ln Keq

Reaction rate definition

Speed of conversion from reactants to products

Rate constant (k)

Frequency of productive collisions

First-order kinetics

Rate depends on one reactant

Second-order kinetics

Rate depends on two reactants

First-order equation

v = k[A]

Second-order equation

v = k[A][B]

Ways to increase rate constant

Increase temperature or decrease activation energy

Temperature effect

Increases molecular collisions and reaction rate

Activation energy effect

Lower energy barrier increases rate

Role of enzymes

Lower activation energy to increase rate

Enzyme mechanism

E + S ⇌ ES ⇌ EP ⇌ E + P

Effect on ΔG°′

Enzymes do not change ΔG°′

Binding energy (ΔGB)

Energy released from enzyme-substrate interactions

Active site definition

Region where substrate binds and reaction occurs

Stable ES problem

If ES is too stable reaction cannot proceed

Solution to stability

Enzyme binds transition state better than substrate

Induced fit definition

Enzyme changes shape upon substrate binding

Purpose of induced fit

Promotes transition state formation

Substrate saturation definition

All enzyme active sites are occupied

Reason rate plateaus

Active sites become saturated at high substrate levels

Initial velocity (V₀)

Reaction rate at time zero

Measurement of V₀

Slope of tangent at t = 0

Effect of substrate concentration

Increasing [S] increases V₀

Pre-steady state

Short phase where ES builds up

Steady state

ES concentration remains constant

Condition for steady state

[S] ≫ [Eₜ]

Michaelis-Menten model

Model describing enzyme kinetics

Mechanism

E + S ⇌ ES → E + P

Assumption 1

Measure initial velocity

Assumption 2

ES concentration is constant

Vmax definition

Maximum reaction velocity at saturation

Units of Vmax

M/sec

Vmax equation

Vmax = k₂[Eₜ]

Km definition

Substrate concentration at half Vmax

Units of Km

Molarity

Km equation

Km = (k₋₁ + k₂)/k₁

Meaning of Km

Reflects enzyme affinity and efficiency

Half-max condition

[S] = Km when v = Vmax/2

Michaelis-Menten equation

v₀ = (Vmax[S])/(Km + [S])

Interpretation of Km

Lower Km = higher affinity

Turnover number (kcat)

Number of reactions per enzyme per second

kcat equation

kcat = Vmax/[Eₜ]

Meaning of kcat

Max catalytic activity of enzyme

Catalytic efficiency definition

kcat/Km

Meaning of catalytic efficiency

Overall enzyme performance

Diffusion limit

10⁸–10⁹ M⁻¹ s⁻¹

Catalytic perfection

Enzyme limited only by diffusion

Environmental factors

Conditions affecting enzyme structure and activity

Effect of pH

Alters Km and/or Vmax

Effect of temperature

Changes enzyme structure and activity

Mutant enzyme Km effect

Higher Km = lower substrate affinity

Interpretation of same Vmax

Maximum rate unchanged but efficiency differs