Enzymes

• Enzyme


  Biomolecules with distinctive 3D structure and employ catalytic mechanisms such as weak interactions, acid-base, covalent & metal ion catalysis
• Active sites

  • Complimentary to transition state of reaction
  • Stronger/additional interactions with transition state lowers activation energy
• Catalytic Mechanisms

  • Acid-base catalysis: give and take protons
  • Covalent catalysis: change reaction paths
  • Metal ion catalysis: use redox cofactors, pKa shifters
• Cofactor


  Small inorganic molecules required by enzymes for activity
• Coenzyme


  More complex molecules that transiently carry functional groups during catalysis of a reaction
• Kinase


  Catalyses the phosphate group transfer from one molecule to another -> move a phosphate from one group to another
• Phosphorylase


  Catalyses the covalent addition of inorganic phosphate (Pi) to a molecule -> more cleaving role
• Phosphatase


  Catalyses the cleavage of a phosphate to yield the dephosphorylated product and Pi
• Dehydrogenase


  Catalyses an oxidation / reduction reaction commonly using NADH/NAD+, NADPH/NADP+ or FADH2/FAD as cofactors
• Mutase


  Catalyses the shift of a phosphate group from one atom to another within the same molecule
• Isomerase


  Catalyses the conversion of one isomer to another
• Hydratase


  Catalyses the addition / removal of water
• Synthase


  Catalyses the synthesis of a product
• Reaction Intermediate

  Stable states found on minima on free energy plot
• Transition State

  Transient species found on maxima on a free energy plot
• Km


  [substrate] at 1/2 Vmax (maximum velocity)
• Michaelis-Menten Model


  The rate equation for a one-substate enzyme-catalysed reaction
• Assumptions of Michaelis-Menten Model

  • ES conversion to E+P irreversible
  • Steady-state conditions
  • [S] >> [Et]
  • [S] >> [P] (initial conditions)
• Lineweaver-Burk Analysis

  X intercept = −1/Km

  Y intercept = 1/Vmax
• Km & Substrate Affinity

  Kd -> how tightly an enzyme binds

  Km -> indication for affinity of the enzyme for substrate (low value corresponds to high affinity)
• Km & Affinity

  Low Km -> high affinity

  High Km -> low affinity
• Turnover Number (k2)


  Number of molecules of substrate converted to product per unit time
• Turnover Number

  High kcat -> fast

  Low kcat -> slow
• Specificity Constant (kcat / Km)

  Rate constant for the conversion of E+S to E+P
• Specificity Constant

  High value -> more efficient use of substrate

  Low value -> less efficient use of substrate
• Irreversible Inhibitors


  Bind covalently to the active site, destroy a functional group essential for enzyme activity, or form a stable noncovalent complex with the enzyme
• Reversible Inhibitors


  Bind reversibly to enzymes and inhibit the enzyme either by competitive, uncompetitive or mixed modes of inhibition
• Competitive Inhibition


  Inhibitor binds to free enzyme (forms EI) at same site as substrate

  When bound -> enzyme has zero activity

  α describes drop in free enzyme concentration [E]
• Uncompetitive Inhibition


  Inhibitor binds to enzyme + substrate complex at allosteric site

  When bound -> enzyme cannot turn over substrate

  α' describes drop in enzyme-substrate concentration [ES]
• Mixed Inhibition


  Inhibitor capable of binding to active site for substrate AND enzyme + substrate complex

  When bound -> enzyme has zero activity

  α and α' used
• Allosteric Enzymes


  Regulate metabolic pathways by changing activity in response to changes in the concentration of molecules around them
• Allosteric Regulation

  
  

  Positive modulators: activate -> stabilise R state -> curve shift to left -> tighter binding

  Negative modulators: inhibit -> stabilise T state -> curve shift to right -> weaker binding
• pH Effect on a-chymotrypsin

  Sharp increase in activity from pH 7 corresponds to changes in kcat

  Below pH 7 -> His57 is protonated & cannot accept proton from Ser195 so kcat ↓

  Above pH 8 -> His57 is all deprotonated so kcat is unchanged

  Above pH 8.5 -> decreased activity -> H+ is lost -> loss of Ile16-Asp194 salt bridge changes hydrophobic pocket where substrate binds -> 1/Km ↓
• For maximum activity: His57 must be unprotonated (>ph 7) and N-ter of the B chain (Ile16) must be protonated (<pH 8.5)
• Catalytic Triad of a-chymotrypsin: serine, histidine, aspartate
• Lineweaver-Burk Plot

  A) Km

  B) Vmax

  C) 1/V0

  D) 1/Vmax

  E) -1/Km

  F) 1/[S]

  
• Enzyme Substrate Reaction

  A) k1

  B) k2

  C) k-1

  
• Michaelis-Menten Model Equation

  A) V0

  B) Vmax[S]

  C) Km + [S]

  D) V0

  E) [S]

  F) Vmax

  G) Km

  
• Km & Substrate Affinity

  A) [ES]

  B) k2 + k-1

  C) k1

  
• Inhibition

  A) Vmax

  B) aKm

  C) Vmax/a'

  D) Km/a'

  E) Vmax/a'

  F) aKm/a'