Binding and recognition of substrates in catalysis Flashcards
(32 cards)
What function do most proteins have?
-the ability to recognize and bind other molecules
-involves the same non-covalent interactions as protein folding
-match shapes to maximize close contact-van der Waals
-match of charged groups or H-bond donors and acceptors
-non-polar patch needs non-polar amino acid
Which molecule binds to polypeptides and finds aromatic amino acids Phe, Tyr and Trp?
-chymotrypsin
-the groove binds to a peptide chain by H-bonds to backbone
-side chain binding pocket is large surrounded by non-polar amino acids
-binding the target positions its peptide bond next to catalytic unit
-binding pocket recognizes the amino acid
-something small can come in and form van-der-waals interaction
What related enzymes are similar to chymotrypsin?
-trypsin and elastase
-different around their side chain binding pockets
-trypsin side chain binding pocket is narrow -charge at end binds Lys, Arg best
-elastase recognizes alanine and glycine the best
-has smaller nonpolar pocket
What makes enzymes catalysts?
-they bind a specific target molecule and speed up a specific chemical reaction
-the target of the enzyme is called its substrate
-many enzymes name ends in -ase
-chemical reaction must be able to occur spontaneously but enzyme will speed it up
Why are uncatalyzed reactions slow?
-reactions depend on random events
-molecules must colide
-they must be in right orientation
-reacting molecules require a threshold energy
-reaction may occur under these conditions but depends on chance
What is the Arrhenius equation?
-rate=p x Z e-(Ea/RT)
-how enzymes speed up reactions
-Z= collision frequency (to speed up)
-p=probability factors that collision leads to reaction related orientation of reactants (to speed up)
-Ea is the activation energy: must be put into a reaction at initial steps to break or distort bonds
-fraction is the molecules temp in kelvin which posses energy Ea
-low Ea or higher T make fraction bigger reaction is favoured
How do enzymes eliminate the randomness of collision?
-random motion of molecules leads to close encounters but few hits between reaction pairs
-enzyme bids substrates in a special pocket known as the active site holding them close together long enough for reaction to proceed (increases collision energy)
-this is the proximity effect -increases Z
What is the orientation effect?
-enzyme binds substrate holding them in the active site so reactive groups are ideally aligned
-increases p
-also applies to enzymes that have only one substrate:
-proximity between substrate reactive groups on enzyme
-ideal alignment of substrate reactive groups on enzyme
-enzyme must hold substrates in the correct orientation
How do enzymes decrease Ea?
-proximity and orientation are physical effects that speed up enzyme reactions
–can also use chemical catalysis to speed up reaction lowering Ea
-can be lowered by finding a better chemical pathway for the reactions involving the functional groups
-Ex: hydrolysis of peptide is very slow because H2O is a very poor nucleophile and a weak acids
-to get faster hydrolysis an acid or base and heating the reaction
-enzymes must speed up reactions at neutral pH and normal temperature
What is nucleophilic catalysis ?
-enzymes can speed up reactions by providing a better nucleophile
Ex: Cys-SH, His-N, Asp or Glu -Coo
rarely Tyr or Ser-OH or Lys :NH2
What is electrophilic
catalysis ?
-an electrophile is an electron-seeking group
-no really good electrophilic amino acids
-enzyme may contain a non-amino acids helper molecule called a prosthetic group as part of its structure
Ex: pyridoxal phospate with its electrophilic aldehyde group (H-C=O)
-binds enzyme catalytic site
initiates reaction by withdrawing electrons from the substrate
What is general acids and general base catalysis?
-general acids: catalysis by an amino acid side chain that donates H+ to the reaction
-General base: catalysis by an amino acid side chains that removes H+ from the reaction
-H+ exchange takes place right at the site of reactions so pH of surrounding is not affected
-gain or loss of one H+ in a small confined volume can have the same effect as strong acid or base
What are the four ways chemcial catalysis can occur?
- Nucleophilic catalysis
- Electrophilic catalysis
- General acid catalysis
4, General base catalysis
-ways the enzyme can get involved in the catalytic process
How do you stabilize the transition state?
-reactions must pass through a transition state to proceed and key atoms may change shape )trigonal planar to tetrahedral or a bond may stretch
-helps lower the Ea
-enzyme provides binding state by stretching bond
-enzyme provides catalytic state that sets up site for state
When is less activation energy needed?
-if the enzyme active site is complementary to the transition state
-differs chemically from the substrate
-substrate pocket–> attractive forces binds it
—> transition state—> is now lower energy and reaction proceeds from transition state to product
How do enzymes increase reactions rate?
-holding reactants close together (Z increase) in the correct orientation (p increase)
2. Chemical catalysis (Ea increase)
a) Nucleophilic
b) Electrophilic
c) Acid
d) Base
3. Stabilizing the transition state (Ea increase)
(Arrhenius as well)
How does the hydrolysis of peptide bonds by chymotrypsin occur?
-chymotrypsin binds weakly to peptide chain upstream of the target amino acid
-the targeted amino acid (Phe, Tyr, Trp) fits into the binding pocket so substrate binds more tightly
-if substrate binding makes a good fit the targeted with the catalytic components (z increase)
How does chymotrypsin catalyze hydrolysis?
-X-CO-NH-Y + H2O—> X-COO- + NH3-Y
How does peptide hydrolysis by water without catalyst?
-water acts as a nucleophile lone pair donates to electron-deficient C
-neutral O does not like to share electrons (electronegative)
-C maintains 8 valence electrons by allowing upper O to take back a bond
-leads to oxyanion transition state where C is now sp3 tetrahedral
-transition state may break down with N as a leaving group (must take electrons lone pair)
-oxyanion O returns the bond to C
-carboxylate C must give up a bond to maintain 8 valence electrons
- if N takes back the excess electrons the peptide bond breaks
What makes the transition state break down?
-by the returning electrons to O
-Carboxylate C may give up the excess bonding electrons to original nucleophilic o
-c-o bond breaks and water is a good leaving group but a poor nucleophile by itself
-reactants are back to the starting point and no net reaction has occurred
How can chymotrypsin do better?
-chymotrypsin uses a nucleophilic group in the enzyme to attack the peptide C=O
-breaks the peptide bond releasing the C-terminal half of the protein
-the N-terminal half remains covalently bonded to the enzyme group (acetyl enzyme intermediate)
-half of the protein chain stays on
2. H20 is brought in to release the N-terminal half and restores the enzyme group to its original state
-graph: substrate, transition state 1, intermediate (solid line) uncatalyzed pathway has dotted line, transition state 2, product
What is the catalytic triad?
-chymotrypsin uses a better nucleophile in the form of the catalytic triad three amino acids that line up side by side in correctly folded chymotrypsin and cooperate for maximum effectiveness
-numbers indicate position in polypeptide sequence
-asp 102-negative charge favours a positive charged partner by influencing His to become positive
-His 57 would be positive if it could capture H +
-can be in both dep/pro state and is mostly neutral
-Ser 195 could give up H+ if it shares a lone pair with a suitable atom
-picks up N from His to become protonated
-its oxygen has 2 lone pairs and is a weak nucleophile
-combined effect makes Ser 195 into a better nucleophile
-always these three amino acids and the number indicate position in polypeptide sequence
What stabilizes the transition state?
-the oxyanion hole
-backbone N-H groups of Gly 193 and Ser 195
What is the first step of nucleophilic attack?
-nucleophilic group in the enzyme attacks the peptide C=O
-His 57 acts as a general base, removing H+ from Ser 195
-Ser 195 becomes a better nucleophile and attacks peptide C=O
-negative charge on Asp102 delocalizes positive charge on His 57
-substrate binding site R= F, Y, W
-C-NH needs to be broken
-curly arrows show the movement of electron pairs
