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Enzymology

Enzymes: core concepts

  • enzymes speed up reactions without being consumed or permanently changed
  • they lower activation energy (energy from initial state to transition-state intermediate)
  • they show specificity to substrate/reactant
  • enzymes have a unique catalytic site (active site)
  • substrates bind to the active site via weak bonds

Models of enzyme–substrate binding

Lock-and-key model

  • substrate “just fits” the active site

Induced-fit model

  • minor conformational changes after initial substrate contact strengthen binding

Allosteric regulation

  • allosteric site: binding site for activators/inhibitors
  • allosteric enzyme: enzyme regulated by allosteric binding

Example:

  • phosphofructokinase-1 (PFK-1)

States:

  • relaxed (binds substrates well)
  • tense (binds substrates weakly)

Apoenzyme vs holoenzyme

  • apoenzyme: inactive enzyme that requires a coenzyme/cofactor
  • holoenzyme: active enzyme with required coenzyme/cofactor bound

Definitions:

  • coenzyme: organic molecule
  • cofactor: metal ion

Isoenzymes

  • differ in properties but catalyze the same reaction

Example (glycolysis-related):

  • hexokinase I, II, III and glucokinase III

  • glucokinase (liver, pancreas): sensor and store glucose; concentration-dependent

Denaturation factors

Heat denaturation

  • bonds break and protein structure is destroyed
  • fever is for moving immune cells (as a note)

pH denaturation

  • disrupts ionic interactions and protein structure

Ionic strength

  • changes in ion concentration affect protein stability and activity

Enzyme classification

Oxidoreductase

  • redox reactions
  • reductant: oxidizing state increases
  • oxidant: oxidizing state decreases

Transferase

  • transfers a functional group from one molecule to another

Hydrolase

  • hydrolysis of substrates

Isomerase

  • structural rearrangement

Lyase

  • cleavage of a carbon–something bond forms a double bond (between carbon and the group that was removed)

Ligase (synthetase)

  • joins two things

Enzyme kinetics (Michaelis–Menten)

Reaction scheme:

  • \(E+S ;\rightleftharpoons^{k_1}_{k^{-1}}; ES ;\to^{k_2}; EP \to E+P\)

Notes:

  • step 1 forward rate: \(k_1\), reverse: \(k^{-1}\)
  • step 2 reverse is negligible
  • step 3 is fast

Key terms:

  • \(v\): reaction rate
  • \(V_{max}\): maximum rate
  • first-order kinetics: rate increases as [S] increases
  • zero-order kinetics: rate does not change as [S] changes

Michaelis constant

  • \(K_m\): substrate concentration where \(v=\frac{1}{2}V_{max}\)
  • affinity is inversely related to \(K_m\)
  • when \([S]\) is above \(K_m\), reaction may no longer be first-order

Michaelis–Menten equation: $$ v=\frac{V_{max}[S]}{K_m+[S]} $$

Assumptions (as listed):

  1. \(E+S\) moves forwards at all time
  2. \([S] \gg [E]\)
  3. \(V_{max}\) is achieved when all enzymes are bound to substrates

Lineweaver–Burk plot

Linear form:

  • \(y=mx+b\)

Definitions:

  • \(y=\frac{1}{v}\)
  • \(x=\frac{1}{[S]}\)
  • \(m=\frac{K_m}{V_{max}}\)
  • \(b=\frac{1}{V_{max}}\)

Inhibitors

Reversible inhibitors (non-covalent)

Example: nucleoside analogs

Competitive inhibition (step 1)

  • occupies active site

  • effects:

  • \(v\) decreases

  • \(V_{max}\) no change
  • \(K_m\) increases

Non-competitive inhibition (step 1/2)

  • binds allosteric site

Example note:

  • binds to cystine and inhibits heme cofactor for hemoglobin

Effects:

  • \(v\) decreases
  • \(V_{max}\) decreases
  • \(K_m\) no change

Uncompetitive inhibition (step 2)

  • binds enzyme–substrate complex

Example:

  • non-nucleoside reverse transcriptase inhibitors (NNRTIs)
  • prevents HIV passing from mother to child

Effects:

  • \(v\) decreases
  • \(V_{max}\) decreases
  • \(K_m\) decreases

Irreversible inhibitors (covalent)

Examples:

  • penicillin
  • nerve gas