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!

Fast

Slow

“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 product

E

ES

Acyl-EE’S2

EP2

Acylationphase

Deacylationphase

The proposed completecatalytic cycle of

chymotrypsin(rate enhancement: 109)A Ping-Pong Mechanism