Two Substrate Reactions
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Transcript of Two Substrate Reactions
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Two Substrate Reactions• Many enzyme reactions involve two or more
substrates. Though the Michaelis-Menten equation was derived from a single substrate to product reaction, it still can be used successfully for more complex reactions (by using kcat).
Random
Ordered
Ping-pong
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Two Substrate Reactions • In random order reactions, the two substrates
do not bind to the enzyme in any given order; it does not matter which binds first or second.
• In ordered reactions, the substrates bind in a defined sequence, S1 first and S2 second.
• These two reactions share a common feature termed a ternary complex, formed between E, ES1, ES2 and ES1S2. In this situation, no product is formed before both substrates bind to form ES1S2.
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Two Substrate Reactions (cont)
• Another possibility is that no ternary complex is formed and the first substrate S1 is converted to product P1 before S2 binds. These types of reactions are termed ping-pong or double displacement reactions.
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The catalytic mechanism of chymotrypsin: a member of the serine protease family; catalyzes the hydrolytic cleavage of peptide bonds adjacent to aromatic amino acid residues (with a rate enhancement
of at least 109).
Principles illustrated:Transition-state stabilization;General acid-base catalysis;
Covalent catalysis.
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Chymotrypsin (and other proteins) are activated via proteolytic cleavage of precursor proteins (zymogens or preproteins).
Many proteases activated this way can be inactivated by inhibitor proteins tightly-bound in the active sites.
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Active chymotrypsin and trypsin are produced from inactive zymogens via proteolytic cleavage, with conformational changes exposing the active sites.
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The catalytically important groups of chymotrypsin were identified by chemical labeling studies
• Organic fluorophosphates such as diisopropylphosphofluoridate (DIPF) irreversibly inactivate chymotrypsin (and other serine proteases) and reacts only with Ser195 (out of the 25 Ser residues).
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A second catalytically important residue, His57, was discovered by affinity labeling with tosyl-L-phenylalanine chloromethylketone (TPCK)
• TPCK alkylates His 57• Inactivation can be inhibited by b-phenylpropionate (competitive inhibitor)• TPCK modification does not occur when chymotrypsin is denatured in urea.
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Rapid initial burst kinetics indicates an acyl-enzyme intermediate• The kinetics of chymotrypsin is
worked out by using artificial substrates (esters), yielding
spectroscopic signals upon cleavage to allow monitoring the rate of
reactions.
Km = 20 mMKcat = 77 s-1
Yellow productColorless substrate
This reaction is far slower than the hydrolysis of peptides!
FastSlow
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“burst” (fast) phase (rapid acylation of all Enzymes leading to release of p-nitrophenol)
Slow phase (enzymes will beable to act again only after a slow deacylation step)
The catalysis of chymotrypsinis biphasic as revealed
by pre-steady state kinetics
Milliseconds after mixing
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Determination of the crystal structure of chymotrypsin (1967) revealed a catalytic triad:
Ser195, His57, Asp102.
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Chymotrypsin: three polypeptide chains linked by multiple disulfide
bonds; a catalytic triad.
His57
Asp102
Ser195
Cleft for binding extended substrates
Trypsin, sharing a 40% identity withchymotrypsin, has a very similar structure.
Active site
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A catalytic triad has been found in all serine proteases: the Ser is thus converted
into a potent nucleophile
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The Peptide Bond has partial (40%) double bond character as a result of resonance of electrons
between the O and N
The hydrolysis ofa peptide bondat neutral pH
without catalysiswill take ~10-1000
years!
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Chymotrypsin (and other serine proteases) acts via a mixture of covalent and general acid-base catalysis to
cleave (not a direct attack of water on the peptide bond!)
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The peptide bond to be cleaved is positioned by the binding of the side chain of an adjacent hydrophobic residue in a special hydrophobic pocket.
Asp102 functions only to orient His57. Formation of the ES complexE
S
ES1
Formation of ES1
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His57 acts as a general base indeprotonating Ser195, the alkoxideion then acts as a nucleophile, attacking the carbonyl carbon.
Ser195 forms a covalent bond with the peptide (acylation) to be cleaved. a trigonal C is turned into a tetrahedral C.The tetrahedral oxyanion intermediate is stabilized by the NHs of Gly193 and Ser195
Preferential binding of the transition state: oxyanion hole stabilization of the
negatively charged tetrahedral intermediate of the transition state.
Pre-acylation
ES1
oxyanion hole
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The amine product is then released from the
active site with the formation of an acyl-enzyme
covalent intermediate.
His57 acts as a general acidin cleaving the peptide bond.
AcylationReleasing of P1
ES1
Acyl-E
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Water (the second substrate) then enters the active site.
Entering ofS2
Acyl-EE’S2
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His57 acts as a general base again, allowing water to attack the acyl-enzyme intermediate,forming another tetrahedraloxyanion intermediate, again stabilized by the NHs of Gly193 and Ser195 (similar to step 2)
Pre-deacylation
E’S2
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His57 acts as a general acidagain in breaking the covalentbond between the enzymeand substrate (deacylation) (similar to Step 3).
Deacylation
EP2
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The second product(an acid) is released from the active site, with the enzyme recoveredto its original state.
Release of P2
Recovered enzyme
EP2
E
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1st substrate
1st product
2nd substrate
2nd productE
ES
Acyl-EE’S2
EP2
Acylationphase
Deacylationphase
The proposed completecatalytic cycle of
chymotrypsin(rate enhancement: 109)A Ping-Pong Mechanism