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
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.
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.
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.
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.
Active chymotrypsin and trypsin are produced from inactive zymogens via proteolytic cleavage, with conformational changes exposing the active sites.
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).
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.
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
“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
Determination of the crystal structure of chymotrypsin (1967) revealed a catalytic triad:
Ser195, His57, Asp102.
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
A catalytic triad has been found in all serine proteases: the Ser is thus converted
into a potent nucleophile
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!
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!)
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
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
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
Water (the second substrate) then enters the active site.
Entering ofS2
Acyl-EE’S2
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
His57 acts as a general acidagain in breaking the covalentbond between the enzymeand substrate (deacylation) (similar to Step 3).
Deacylation
EP2
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
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