Figure 11-13b

Binding features that promote catalysis

• co-localization and proper orientation of reacting groups

• involving different molecules or just a single molecule

• binding energy is used to pay the entropic cost of bringing reacting groups into the right positions and orientations

• stabilization of (and thereby population of) particular configurations of molecules suitable for bond formation/breakage

• electrostatic contributions; stabilization of the charged states that develop during a reaction

Figure 11-13b

An example of the importance of proximity effects

slow reaction

fast reaction

Figure 11-13b

Understanding catalysis as a lowering of the transition state free energy

• articulated by Linus Pauling• recall that providing something that binds a molecule

tightly effectively lowers its free energy

If the enzymes binding site is most complementary to the transition state (and so lowers the energy of the transition state more than the substrate/products), then the activation free energy is lowered, leading to rate enhancement.

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The importance of transition state theory

• helps explain the mechanisms of catalysis• provides a strategy for designing inhibitors against

various enzymes• imagine what the (unstable) t.s. for the reaction must

look like• design a (stable) organic mimic that looks similar; this

should bind very more tightly to the enzyme than the substrate, and act as a potent inhibitor

• an important strategy in drug design

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Example of an inhibitor based on a transition state analogue

reaction catalyzed by the enzyme proline racemace

transition state analogues sharing some features with the presumed transition state

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Mechanism of a well-studied enzyme: lysozyme• hydrolyzes (14) polysaccharides linkages in the

bacterial cell wall• resembles an acid catalyzed mechanism in solution• Glu 35 acts as an acid, donating proton to leaving group• carbocation stabilized by

• oxonium resonance, promoted by binding the sugar ring in a half-chair configuration

• then by covalent bond to Asp 52• 2nd half of the reaction is the reverse of the first

• H20 displaces the leaving sugar• Glu 35 now acts as a base to accept proton from

H20, which becomes the attacking group when the Asp 52 leaves

• note that ‘double displacement’ leads to retention of the configuration at the anomeric carbon

Figure 11-16

substrate

enzyme. note the active site has sub-sites for binding multiple parts of the sustrate

Figure 11-19

A planar arrangement of atoms attached to the ring oxygen is required for stabilization of the carbocation that develops at the anomeric carbon when the leaving group leaves. Such a planar configuration is provided by the half-chair ring conformation, which is how the active site binds the substrate.

Figure 11-21

Lysozyme mechanism

Figure 11-21 part 1

Lysozyme mechanism

Figure 11-21 part 2

Lysozyme mechanism

Figure 11-21 part 3

Lysozyme mechanism

Figure 11-21 part 4

Lysozyme mechanism

Figure 11-21 part 5

Lysozyme mechanism

Figure 11-22

Lysozyme transition state and t.s. analog

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Mechanism of a well-studied family of enzymes: serine proteases

• proteases hydrolyze other proteins

• widespread• use a serine nucleophile to

attack substrate carbonyl• because the serine

nucleophiles in these enzymes are so nucleophilic, they can be ‘killed’ (i.e. irreversibly modified by a covalent bond) by ‘suicide inhibitors’

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Mechanism of a well-studied family of enzymes: serine proteases

• specificities of different serine proteases vary (e.g. trypsin, chymotrypsin, etc.)

• specificity pockets in the binding site for recognizing certain amino acids in the substrate proteins

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Mechanism of a well-studied family of enzymes: serine proteases

• Mechanism:• serine is promoted as a nucleophile by

deprotonation by nearby histidine side chain (whose resulting positive charge is stabilized by a nearby aspartate). This is the famous ‘catalytic triad’

• departure of C-terminal fragment of substrate• a covalent intermediate is formed in which the N-

terminal fragment of the substrate is attached to the enzyme as an acyl-enzyme intermediate

• 2nd step is a direct repeat, but with H20 replacing the serine as the nucleophile

Figure 11-26

Serine proteases: the catalytic triad

note that the participating residues are coming from different parts of the enzyme sequence

Figure 11-28

Similar catalytic triads have been discovered in apparently unrelated enzymes, having different overall three-dimensional structures and different orderings of the triad residues, suggesting that this particularly useful arrangement of catalytic residues has evolved independently multiple times (i.e. ‘convergent evolution’)

Figure 11-29

Serine protease mechanism

Figure 11-29 part 1

Serine protease mechanism

Figure 11-29 part 2

Serine protease mechanism

Figure 11-29 part 3

Serine protease mechanism

Figure 11-29 part 4

Serine protease mechanism

Figure 11-29 part 5

Serine protease mechanism

Figure 11-30a

Serine proteases: features for transition state stabilization

• ‘oxyanion hole’ to stabilize negative charge on tetrahedral intermediate

• H-bonds to stabilize distorted protein backbone

Box 11-4c

One biologically important use of proteases: to cleave inactive proenzymes (or zymogens) to produce the mature, active form of an enzyme

In some cases, such cleavages act one after another on a series of enzymes. This leads to a ‘cascade’, which can produce a geometric explosion of enzymatic activity (which may be needed in response to catastrophic events). The well-known blood clotting cascade is shown.