VL 4 (Silke Leimkühler)

Question 1

Catalysts: Definition

Answer 1

A catalyst increases the rate of the reaction without being consumed and without changing the chemical euqilibrium of the reactions.
- Acceleration of the reaction by reducing the activation energy

Question 2

Gibbs free energy is a useful thermodynamik function for understanding enzymes

Answer 2

The free-energy change provides information about sponateity but not the rate of a reaction.

• spontaneous reaction: ΔG < 0 (exergonic reaction)
• no spontaneous reaction: ΔG > 0 (endergonic reaction)
• equilibrium: ΔG=0
  →no net change in reactant/product amount

ΔG of a reaction:
* depends only on ΔG difference between reactants-products + independet of how reaction occurs
* provides no information about reaction rate

transition state:
* designated by double dagger: ‡
* energy required to form the transition state from substrate = activation energy

G = Gibbs free Energie
DG = Gibbs free energy change
DG0 = standard free energy change (25°C, 1M, 1 atm)
DG‡ = activation energy

Question 3

Binding Energy between Enzymes and substrate

Answer 3

Binding energy:
* between enzymes and substrate is important for catalysis
* is the free energy released upon interaction of the enzyme and substrate
* is greatest when the enzyme interacts with the transition state, thus
facilitating the formation of the transition state

Question 4

What does common catalytic reactions include?

Answer 4

1. acid-base catalysis: a molecule other than H2O donates/accepts a H+
2. Electrostatic catalysis: charge stabilization by a charge from amino acid at active site
3. Metal ion catalysis: metal ions serve as electrophilic catalysts
4. Catalysis by approximation: E brings two S together in an orientation that facilitates catalysis energy
5. Covalent catalysis: active site with Nu- that is briefly covalently modified

Question 5

Explain Acid-Base catalysis

Answer 5

• Asp, Glu, His, Lys, Cys, Tyr –> Amino acid side chains that can act as acid-base catalysts
• catalysts often sesitive to pH changes
• acid transfers H+
• base abstracts H+
• aa-side chains: a/b-catalysts
• governed (regiert/verwaltet) by sidechain pKa ́s→pH-dependence
• pH-rate profiles
  →distinguish betweena/b catalysis
  →identification of participating catalytic residues (mutagenesis)
• example: RNase A

Question 6

Explain electrostatic catalysis

Answer 6

Asp, Glu, His, Lys, Arg –> Amino acid side chains thart participate in electrostatic catalysis

• No stabilization of charged transition state by acid-base catalyst
  →charge can be neutralized by oppositely charged group from catalyst (active site of enzyme)
• S-binding to active site in H2O absence
• enhancement of electrostatic interaction in nonpolar environment of E interior
• H2O excluded from active sites, exception: catalytically needed
  →low dielectric environment where electrostatic interactions are stronger
• E-transition state interactions involving charge stabilization are enhanced
• example: orotidine 5 ́-monophosphate decarboxylase

Question 7

Explain Metal ion catalysis

Answer 7

13 different Metall-Ions were identified with roles in biologic reactions:
Mg, Zn, Fe, Mn, Ca, Co, Mo, W, Cu, Na, K, Ni, V

• 1/3 of all Enzymes contain metals
• Metal ions promote reactions + protein folding

Example: carbonic anhydrase
* CO2 = end product of aerobic metabolism
* Lungs: bicarbonate→CO2→exhaled
* 7 homologous genes encoding Enzyme
* Zn2+
–> bound to 3 His, one binding site binds H2O
–> CO2 binding site adjacent
–> Lowers H2O pKa to 7, generating the nucleophile OH-
–> OH- attacks CO2 converting into bicarbonate
* max. rate of CO2 hydration attained at pH 8
* acitivity falls with pH drop
* titration curve suggests that active site component with pKa = 7 is required

Question 8

Explain catalysis by approximation

Answer 8

Enzyme brings two Substrates together in an orientation that facilitates catalysis

a) S approximation (up to 10x reaction rate acceleration)
b) S orientation (up to 100x reaction rate acceleration)
c) hindrance of rotation-, translation freedom (entropic traps)

Example: ribosome

Question 9

Explain covalent catalysis

Answer 9

Covalent catalysis can be divided into 3 steps
1. Nucleophile reaction of E&S →formation: covalent bond (initiated by e- rich group in active site)
2. e- abstraction by electrophilic groups
3. covalent bond cleavage + reversal of 1st reaction step

• Covalent bond is formed between E and its S during formation of transition state
  –> initiated by an electron rich gropu in the active site
• Covalent catalysis is a two-part reaction process conatining two energy barriers in the reaction coordinate diagram

Question 10

Protease and catayltic triade

Answer 10

Serine, Histidin and Aspartate –> catalytic triad
* Histidine removes a proton from serine 195
* generating a highly reactive alkoxide ion.
* The alkoxide ion attacks the peptide bond of the substrate.
* Aspartate orients the histidine and renders it a better proton acceptor
* catalytic triad (dissected by site-directed mutagenesis):

Reaction:
1. S-binding
2. Nucleophile attack of O-atom (serine) on carbonyl group of peptide bond
(→Ser deprotonated, O- group (deprotonation assisted by Asp102 (deprotonated)).
3. H+ transfer from +-charged His57→NH2 group→disassembly: tetrahedral intermediate.
4. Release of amine component →
1st step: acylation completed
5. H2O attachment
6. Nucleophile H2O attack on acyl enzyme intermediate (H2O deprotonation by deprotonated His57 → OH- - ion → Nucleophile attack of OH- -ion on carbonyl C atom of acyl group → tetrahedral intermediate)
7. Decomposition: tetrahedral intermediate
8. Release: carboxylic acid component

Oxyanion hole:
- Function: stabilizes tetrahedral intermediate