Lecture #5 Catalytic and Regulatory Strategies Mechanistic studies seek to answer the question, &quot;How do enzymes achieve catalysis?&quot; The first slide lists a number of ways in which enzymes could possibly speed a reaction. 1. Enzymes bind substrates close to each other and in the correct orientation so that reaction may occur without the need for a random collision between reactants. 2. An enzyme can act as an acid or base (or both) which eliminates the need for extremes in pH for catalysis to occur. 3. Charged groups on the enzyme can stabilize charged transition state intermediates thereby lowering the energy of activation. 4. Enzyme side chains can function as nucleophilic or electrophilic reagents. 5. Changes in shape of the active site can induce strain in the substrate which promotes rapid reaction. Proximity Effects: The chemical example of anhydride formation shows how having two reacting groups in proximity to each other can facilitate reaction Other Effects (not shown) There are a variety of way in which an enzyme can facilitate catalysis. They are shown here fyi. Many of these catalytic strategies will be seen in the mechanism used by the enzyme, Chymotrypsin (see below) -Acid-Base Catalysis: In this example the enzyme can act as a proton donor (Acid) or proton acceptor (Base) to promote rapid reaction. -Electrostatic effects: The formation of a positively charged covalent intermediate (Schiff Base) between enzyme and substrate promotes the movement of electrons that is needed for the decarboxylation of acetoacetate. That is, the enzyme provides a alternate pathway of reaction with a lower energy of activation. -Nucleophilic Catalysis: In hydrolysis reactions there is often a <a href="/keyword/nucleophilic-attack/" >nucleophilic attack</a> of a free hydroxyl group on a carbonyl carbon. In certain serine proteases an active site serine (hydroxyl) group substitutes for a free hydroxyl group resulting in a covalent enzyme-substrate (acylenzyme ester) intermediate. Since the serine hydroxyl is always present in the active site of the enzyme, the rate of reaction in increased. -Entropy Loss: Binding to the enzyme reduces random tumbling and stretching of the substrate. This is a low-entropy state that favors reaction of the ES complex. -Desolvation: Substrates often have water tightly bound to their surface. Binding to the enzyme can release this water and make the substrate more reactive. -Electrostatic Destabilization: If repulsive charges occur between substrate and enzyme and these repulsive forces can be relieved during reaction, the reaction rate may be speeded. Slide 4 shows the hydrolysis of an amide bond. This is the type of reaction catalyzed by the enzyme chymotrypsin. It is a hydrolytic reaction in which the elements of water combine with the amide to produce and acid anion and an amine cation. The enzyme chymotrypsin serves as a well-studied prototype for considering enzyme mechanisms. Chymotrypsin exhibits a number of interesting features including zymogen activation, an active site serine residue, formation of a covalent acyl-enzyme intermediate, acid base catalysis, and a charge relay network (catalytic triad).Chymotrypsin cleaves peptide (amide) bonds on the amino side of bulky <a href="/keyword/amino-acid-residues/" >amino <a href="/keyword/acid-residues/" >acid residues</a> </a> such as phenylalanine and methionine. Chymotrypsin is a serine protease. Its active site has a uniquely active serine hydroxyl group that can lose a proton and become anionic during the course catalysis. That property makes the active site serine residue highly susceptible to reaction with the reagent DIFP. This reaction produces a covalent product and inactivates the enzyme. The assay of chymotrypsin is accomplished using an artificial substrate that mimics peptides, but whose hydrolysis is easily measured. When the N-acetyl-L-phenylalanine p-nitrophenyl ester is hydrolyzed, a colored product, p-nitrophenylate is released. The rapid kinetic analysis of p-nitrophenylate release shows that there is a burst phase during the first round of hydrolysis followed by a steady state phase which last through many rounds of cleavage. This suggests that the deacylation of the enzyme is the rate limiting step in hydrolysis of the N-acetyl-L-phenylalanine p-nitrophenyl ester. Slide 9 gives a generalized scheme for the enzymatic hydrolysis of amides or esters in reactions involving active site serine residues. There is an acylation phase in which the alcohol or amine is released and the active site serine is acylated. That is followed by a deacylation phase in which the carboxylic acid component is released from the enzyme. The intermediate in this process is an acylenzyme intermediate in which the carboxylic acid portion of the substrate is covalently bound to the surface of the enzyme. The active site of chymotrypsin contains a catalytic triad, containing the amino acids His 57, Asp 102 and Ser 195, that is especially important in catalylsis. Hydrogen ions and electrons are passed back and forth between these three amino acids which has the net effect of stabilizing the serine hydroxyl group in the negative anionic state. Note that these active site amino acids come from <a href="/keyword/amino-acid-residues/" >amino <a href="/keyword/acid-residues/" >acid residues</a> </a> that are spatially separated in the primary sequence of the enzyme. Slide 11 shows the overall mechanism of chymotrypsin. Hydrolysis of an amide substrate involves the participation of the catalytic triad, the formation of a covalent acylenzyme intermediate, and the transient existence of various tetrahedral transition state intermediates. In step 1 of the reaction there is a nucleophylic attack by the active site serine on the carbonyl group of the amide substrate. In step 2 the first tetrahedral intermediate is formed. This intermediate is unstable because its anionic character (negative charge on the oxygen). That anionic intermediate is stabilized by charged groups in the &quot;anionic hole&quot; in the enzymes active site. This stabilization lowers the energy of activation and speeds the rate of reaction. The enzyme is providing an alternate reaction mechanism that has a reduced energy of activation. In step 3 the tetrahedral intermediate breaks down, cleaving the amine product from the rest of the substrate. The portion of the molecule that remains covalently bound to the active site serine corresponds to the carboxylic acid part of the substrate. This is the acylenzyme intermediate which is much more stable than the anionic intermediates. Under certain conditions serine proteases can be isolated with the acylenzyme intermediate covalently attached to the enzyme active site. In step 4 the amine product picks up a proton from the active site histidine and diffuses away. In this reaction the histidine is acting as an acid (proton donor) In step 5 there is a nucleophylic attack of a hydroxyl ion on the acylenzyme intermediate. The production of the hydroxyl ion from water in the active site is promoted by the active site histidine which acts as a base (proton acceptor) to remove the proton from the water molecule. In step 6 a second tetrahedral intermediate is formed. Similar to the chemistry of step 2, this intermediate is unstable because its anionic character (negative charge on the oxygen). That anionic intermediate is again stabilized by charged groups in the &quot;anionic hole&quot; in the enzymes active site. This is a second transition state intermediate whose energy of activation is reduced by interaction with the active site of the enzyme. In step 7 second tetrahedral intermediate is attacked by a proton conveniently provided by the active site histidine residue. The anionic oxygen is converted to a carbonyl and the attachment of the intermediate to the active site serine is broken. In step 8 the carboxylate product diffuses away and the enzyme reverts to its original state. Figure 9.9 show how the tetrahedral anionic intermediate is stabilized...