Protein binding & recognition: Chemical basis of enzyme catalysis Flashcards
What does the function of most proteins involve?
Function of most proteins involves ability to recognize and bind other molecules
Involves the same non-covalent interactions as protein folding
- Non-polar patches on surface of target bind by hydrophobic effect
- Match shapes to maximize close contact – van der Waals effect
- Match of charged groups or H-bond donors and acceptors
Protein-protein interactions are one of the fundamental properties of what?
How does this relate to enzymes and antibodies?
Protein-protein interactions are one of the fundamental properties of living processes
- Proteins that form quaternary structure recognize and bind their partner proteins
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Enzymes recognize and bind their specific target proteins, and catalyse reactions on them,
- e.g. phosphorylation
- Antibodies bind and identify foreign molecules e.g. from bacteria and viruses, and tag them for attack
Chymotrypsin binds to polypeptides and finds which amino acids?
Chymotrypsin binds to polypeptides and finds aromatic amino acids Phe, Tyr and Trp
- Groove in chymotrypsin binds a peptide chain by H- bonds to backbone
- Side chain binding pocket is large, and surrounded by non-polar amino acids of the chymotrypsin
- Phe, Tyr, Trp fit best
- Binding the target positions its peptide bond next to catalytic unit
Which related enzymes are similar to chymotrypsin? How do they differ?
Related enzymes trypsin and elastase are similar to chymotrypsin except around their side chain binding pockets
Similar primary structure and tertiary structures
What are enzymes?
Why are uncatalyzed reactions slow?
- Enzymes bind a specific target molecule (or molecules) and catalyze a specific chemical reaction.
- The target of the enzyme is called its substrate
- Many enzyme names end in –ase, e.g. ribonuclease
- The chemical reaction must be able to occur spontaneously, but enzyme speeds it up by factor of 106 and 1017, typically 1010-fold
Why are uncatalyzed reactions slow?
- Without catalyst, reactions depend on random events
- molecules must collide
- they must be in right orientation
- reacting molecules require a threshold energy
If these conditions are met, reaction may occur, but depends on chance
How do enzymes speed up reactions?
The Arrhenius equation: rate = pZe-(Ea/RT)
- Z is the collision frequency
- p is the probability factor, probability that collision leads to reaction – related to orientation of reactants
- Ea is the activation energy; energy must be put into a reaction at initial steps, to break or distort bonds
- e–Ea/RT is the fraction of molecules at temp T (in Kelvin) which possess energy Ea
Low Ea or higher T make fraction bigger, so reaction is favoured
What do enzymes eliminate?
Enzymes eliminate the randomness of collision
- Random motion of molecules leads to close encounters but few hits between reacting pairs
- Enzyme binds substrates in a special pocket known as the active sites, holding them close together long enough for reaction to proceed
- This is the proximity effect– increases Z
How do enzymes hold substrates in the correct orientation?
Enzymes hold substrates in the correct orientation
- Two molecules may meet by random collision, but reactive groups may not be properly lined up for reaction
- Enzyme binds substrates, holding them in the active site so reactive groups are ideally aligned
- This is the orientation effect– increases p
Proximity and Orientation
What does randomness relate to?
- Enzymes eliminate randomness of reaction processes
- Randomness relates to entropy
- enzymes decrease activation entropy of reaction
- calculation of entropy shows that each effect can speed-up a reaction by 103 - 105-fold, maximum of 1010-fold when combined
- Also applies to enzymes that have only one substrate
- Proximity between substrate and reactive groups on enzyme
- Ideal alignment of substrate and reactive groups of enzyme
What do enzymes decrease?
Enzymes decrease Ea
- Proximity and orientation are physical effects that speed up enzyme reactions
- Enzymes can also use chemical catalyst to speed up reaction by lowering Ea
Ea can be lowered by finding a better chemical pathway for the reaction, involving reactive groups on the enzyme
X-CO-NH-Y + H2O → X-COO- + +NH3-Y
- Reaction shown – hydrolysis of peptide - is very slow because H2O is a very poor nucleophile, and very weak acid
- A chemist would use acid or base, and heat the reaction to get faster hydrolysis
- Cells exist close to neutral pH, and at relatively low, fixed temperature
- Enzymes must speed up reactions at neutral pH and normal temperature
What is a nucleophilic and electrophilic catalyst?
- Nucleophilic catalysis: enzymes can speed up reactions by providing a better nucleophile
– e.g. Cys-SH, His-N:, Asp or Glu-COÖ–,
– more rarely Tyr or Ser -ÖH or Lys :NH2
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Electrophilic catalysis:
- an electrophile is an electron-seeking group
- no really good electrophilic amino acids
- enzyme may contain a non-amino acid helper molecule
- called a prosthetic group, as part of its structure
- e.g. pyridoxal phosphate (cofactor or coenzyme) with its electrophilic aldehyde group
- binds at enzyme catalytic site, initiates reaction by withdrawing electrons from the substrate
What are general acid and general base cataclysts?
- General acid: catalysis by an amino acid side chain that donates H+ to the reaction
- General base: catalysis by an amino acid side chain that removes H+ from the reaction
- H+ exchange takes place right at the site of reaction, so pH of surroundings is not affected
- Gain or loss of one H+ in a small confined volume can have same effect as strong acid or base
What are the four chemical catalysts?
- Nucleophilic catalysis
- Electrophilic catalysis
- General acid catalysis
- General base catalysis
* these mechanisms can each contribute about 100-fold increase in reaction rate, and work together to lower Ea
What must reactions pass thrpugh to proceed?
How can the enzyme help?
Stabilizing the transition state
- Reactions must pass through a transition state to proceed, and key atoms may change shape, e.g. trigonal planar to tetrahedral, or a bond may stretch
- The enzyme can help by binding the substrate in the ideal shape for the transition state
When is less activation energy needed?
Less activation energy is needed if the enzyme active site is complementary to the transition state
