Enzymes

• Enzymes are biological catalysts → help reactions proceed faster
• Lower activation energy → increase rate
• Make reactions more KINETICALLY favorable
• Not changed/consumed
• Don’t alter equilibrium constant
• Don’t change $\Delta G$
• pH and temperature sensitive
• Usually specific (specific reactions/substrates)
  • Urease only breaks down urea
  • Chymotrypsin catalyzes peptide bonds between AAs with aromatic rings
• Enzymes bind substrates at the transition state

Oxidoreductases

• Catalyze redox reactions (transfer of e⁻)
• Often use cofactors like NAD⁺ or NADP⁺
• Electron donor = reductant
• Electron acceptor = oxidant
• Oxidase: oxygen is final electron acceptor
• Dehydrogenases and reductases are oxidoreductases

Transferases

• Transfer functional groups
• Aminotransferases convert aspartate + α-ketoglutarate → glutamate + OAA
• Kinases are transferases

Hydrolases

• Break compounds using addition of water
• Phosphatase cleaves phosphate group
• Peptidase, nuclease, lipase all break down stuff

Lyases

• Cleave one molecule into 2 products
• Can catalyze reverse reaction (synthesis of 2)
  • Then called synthases
• Often form cyclic compounds or double bonds
  • Example: ATP → cAMP

Isomerases

• Rearrangement of bonds within a molecule
• Convert between stereoisomers and constitutional isomers

Ligases

• Addition/synthesis reactions
• Often require ATP
• Nucleic acid synthesis and repair

Impact on activation energy

• Endergonic reaction requires energy: $\Delta G > 0$
• Exergonic reaction releases energy: $\Delta G < 0$
• Enzymes increase rate at which equilibrium is reached
• Lower activation energy
• Reverse is energetically very unfavorable

• Enzyme binds substrate → forms ES complex
• Active site is where substrate is held
• Bonding stabilizes active site

Lock-and-key theory

• Active site is already shaped for substrate
• No structural change needed

Induced fit model

• Active site changes conformation to fit substrate
• Conformational change is endergonic; return to normal is exergonic
• When substrate is present, active site becomes ready
• Wrong substrate won’t induce fit

Cofactors and coenzymes

• Small enough to bind at active site
• Participate directly in catalysis
• Carry charge via ionization/protonation/deprotonation
• Apoenzyme: enzyme without cofactors
• Holoenzyme: enzyme with cofactors
• Prosthetic groups: tightly bound cofactors
• Cofactors: inorganic molecules/ions (ingested as minerals)
• Coenzymes: small organic groups (vitamins/derivatives)
  • NAD⁺, FAD, CoA
• Water-soluble vitamins: B vitamins and ascorbic acid (vitamin C)
  • Important coenzymes are excreted → must be replenished
• Reactions can require multiple cofactors/enzymes

• As substrate increases, rate increases then levels off as active sites fill
• At saturation, rate = $V_{max}$
• To increase $V_{max}$, increase enzyme concentration $[E]$

Michaelis–Menten equation

• Describes rate dependence on $[E]$ and $[S]$
• ES forms at $k_1$
• ES dissociates at $k_{-1}$ or forms $E + P$ at $k_\text{cat}$
• $V = \dfrac{V_{max} [S]}{K_m + [S]}$
• When $V = \dfrac{1}{2}V_{max}$, then $K_m = [S]$
  • If $[S] = K_m$, rate is half of $V_{max}$
• $K_m$ = substrate concentration at “half saturation” (Michaelis constant)
• Used to compare enzymes
• $K_m$ can reflect affinity:
  • Higher $K_m$ = lower affinity (need more substrate to half-saturate)
• If $[S] \gg K_m$, rate ≈ $V_{max}$
• Relationship is usually a parabola
• Low $[S]$: small $\Delta [S]$ greatly changes rate
• High $[S]$: $\Delta [S]$ doesn’t change rate much

Linking $V_{max}$ to $k_\text{cat}$

• $V_{max} = [E]k_\text{cat}$
• $k_\text{cat}$ = substrate molecules turned over per enzyme per second
• Most enzymes: $10^1$ to $10^3$
• $V = \dfrac{k_\text{cat}[E][S]}{K_m + [S]}$

$k_\text{cat}/K_m$ ratio

• Catalytic efficiency
• Larger $k_\text{cat}$ and smaller $K_m$ → higher efficiency

Lineweaver–Burk plots

• Double reciprocal form of Michaelis–Menten
• x-intercept: $-\dfrac{1}{K_m}$
• y-intercept: $\dfrac{1}{V_{max}}$
• Used to determine inhibition type

Cooperativity

• Some enzymes show sigmoidal kinetics due to cooperativity
• Often multiple subunits/active sites
• Enzyme exists in:
  • T state (tense, low affinity)
  • R state (relaxed, high affinity)
• Substrate binding shifts T → R
• Increases odds of binding at other subunits
• Party analogy: as more people arrive the party seems better; as more leave, more want to leave
• Affinity with 2 subunits bound > with 1 bound, but < with 3 bound

Hill coefficient

• Indicates cooperativity:
  • 1 = positive cooperativity (binding increases future affinity)
  • <1 = negative cooperativity
  • =1 = no cooperativity

Temperature

• Reaction rate doubles for every 10°C increase until optimal temp
• Past optimal temp → enzymes denature
• Siamese cats: tyrosinase for pigmentation
  • Cooler areas (tail/feet/ears/face) → enzyme active → darker fur
  • Without enzyme, peak temp is higher

pH

• Optimal body pH ~7.4
• Enzymes have different optimal pH:
  • Pepsin ~2
  • Pancreatic enzymes ~8.5

Salinity

• Salt concentration can affect enzyme activity in vitro
• Can disrupt H-bonds and ionic bonds
• Causes conformational change
• Can cause denaturation

Feedback regulation

• Negative feedback: enough product → stop making more

Reversible inhibition

• Competitive inhibition
  • Inhibitor blocks active site → substrate can’t access
  • Add more substrate to overcome
  • $V_{max}$ unchanged
  • $K_m$ increases
• Noncompetitive inhibition
  • Inhibitor binds allosteric site → conformational change
  • Can’t be overcome by adding substrate
  • $V_{max}$ decreases
  • $K_m$ unchanged
• Mixed inhibition
  • Inhibitor binds E or ES with different affinities
  • Binds allosteric site
  • $K_m$ changes depending on preference:
    • If binds E → $K_m$ increases (affinity ↓)
    • If binds ES → $K_m$ decreases (affinity ↑)
  • $V_{max}$ always decreases
  • Lines intersect not on an axis
• Uncompetitive inhibition
  • Binds only ES complex
  • Locks substrate in enzyme → prevents release
  • Increases affinity between enzyme and substrate
  • Must bind allosteric site
  • Lowers both $K_m$ and $V_{max}$

Irreversible inhibition

• Active site unavailable for a long time
• Can’t reverse
• Example: aspirin irreversibly modifies COX-1 → can’t make prostaglandins
  • Must synthesize new COX-1 to restore function
• Prime drug mechanism

Regulated enzymes

• Allosteric enzymes
  • Multiple binding sites
  • Switch between active/inactive forms
  • Activators or inhibitors bind → conformational shift
  • Plots often sigmoidal
• Covalently modified enzymes
  • Phosphorylation or dephosphorylation
  • Can’t predict effect without experiments
  • Glycosylation is another modification
    • Tags for transport or modifies protein activity
• Zymogens
  • Inactive enzymes that require modification/removal of regulatory domain to expose active site
  • Often end in -ogen (e.g., trypsinogen)