VL 3 (Silke Leimkühler) Flashcards
Enzymes in general
- Enzymes are powerful and highly specific catalysts
- Two models have been proposed to explai how an enzyme binds it substrate:
- The Lock and key model
- The induced-fit model
Free energy vs. Reaction progress
What are Enzyme kinetics
- study of chem. reactions that are catalyzed by enzymes
- reaction rate measured (enzyme-catalyzed reaction rates at different c[S] with constant c[E])
- effects of varried reaction conditions are investigated
Michelis Menten kinetics and its parameters
- turnover number: kcat = Vmax/[E]t
- number of substrate molecules converted into product (S→P) per second at a single catalytic site of an enzyme (min-1, sec-1)
- Michaelis-Menten Konstant: KM
- = relative enzyme affinity towards substrate (M)
- equivalent to c[S] that an enzyme turns over at one half of its maximum rate
- Catalytic Effciency: kcat/KM
- measure how efficiently an enzyme converts S→P (sec-1M-1)
Example: carbonic anhydrase
Michaelis-Menten Equation
- describes initial reaction velocity (V0) as a function of c[S]
- v0 = 1⁄2 Vmax, KM = c[S]→KM is c[S] that yields 1⁄2
What is the linearization after Eadie-Scatchard?
What is lineraization after Hanes-Woolf?
Linearization after Cornish-Bowden/Eisenthal?
Km and Vmax
Km:
Km= (k-1 + k2) / (k1)
* ~ c[S] of the enzyme in vivo
* ~ dissociation constant of ES-complex if k-1»k2
Vmax:
Vmax= k2 [E]T
* known enzyme concentration, [E]t:
* k2=kcat
What is Cleland´s nomenclature system for enzymatic reactions?
Multi-substrate reactions
- substrate numbers
–> bi-/tri-/tetra-substrate reaction - follow Michaelis-Menten equation
- classes
–> sequential: ternary complex formation (E + both substrates)
▪ random: S-binding in random order (either substrate A/B bind enzyme first; either product P/Q is released first)
▪ ordered: order of S-binding (first S binds before second; first P released before second)
–> double-displacement/Ping-Pong: formation of a substituted enzyme intermediate
▪ substrate A binds enzyme E→product P + modified enzyme E ́
▪ substrate B binds modified enzyme E ́→product Q + enzyme E
What is a sequential reaction?
Bi-substrate kinetic?
Inhibition of enzymes
Reversible Inhibition is characterized by a rapid dissociation of the enzyme-inhibitor complex
4 common types of reversible inhibition:
1. Competitive inhibition
2. Uncompetitive inhibition
3. Non-competitive inhibition
4. Mixed-type Inhibition
What is competitive Inhibition?
▪ structurally similar I to S
▪ bind free enzyme E→no S-binding
▪ no ES-binding
▪ dissociation constant Ki = [E][I]/[EI]
▪ higher c[S]→inhibition overcome→Vmax unchanged
▪ Km in I-presence (Kmapp) is increased: Kmapp = Km(1+[I]/Ki)
What is non-competitive Inhibition?
▪ I binds either E or ES→prevents P-formation
▪ Ki, Ki ́ = identical
▪ Vmax in I-presence (Vmaxapp) lower (can ́t be overcome by higher c[S])→Vmaxapp = Vmax/(1+[I]/Ki)
▪ Km unchanged
What is uncompetitive inhibition?
▪ I binds ES→ESI→no P
▪ Vmaxapp lower (can ́t be overcome by higher c[S])
▪ Km lower
What is mixed-type inhibition?
▪ I binds either E or ES
▪ Ki, Ki ́ = different
Differentiating Bi-Bi mechanism by product inhibition
competitive inhibition –> Substrate and inhibitor competitively bind to the same site of the enzyme
