enzymes 2

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Strategies in Enzyme Catalysis: 

Strategies in Enzyme Catalysis As stated earlier, the role of a catalyst is to decrease the energy of activation of a reaction–the energy necessary to attain the transition state. Several themes recur in enzyme catalysis. Catalysis by approximation General acid, general base catalysis Catalysis by electrostatic effects Covalent catalyis (nucleophilic or electrophilic) Catalysis by strain or distortion For most enzymes, more than one of these strategies are used concomitantly

Catalysis by Approximation: 

Catalysis by Approximation The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. If DG‡ is the change in free energy between the ground state and the transition state, then DG‡=DH‡–tDS‡. In solution, the transition state would be significantly more ordered than the ground state, and DS‡ would therefore be negative. The formation of a transition state is accompanied by losses in translational entropy as well as rotational entropy. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. Therefore, there is no loss in translational or rotational energy in going to the transition state. This is paid for by binding energy.

Entropy and Catalysis: 

Entropy and Catalysis

Orientation Effects: 

In the non-enzymatic lactonization reaction shown below, the relative rate when R = CH3 is 3.4 x1011 times that when R = H. What is the explanation? Orientation Effects

Catalysis by Approximation: 

Catalysis by Approximation In order for a reaction to take place between two molecules, the molecules must first find each other. This is why the rate of a reaction is dependent upon the concentrations of the reactants, since there is a higher probability that two molecules will collide at high concentrations. As an example, look at the hydrolysis of paranitrophenyl ester again catalyzed by imidazole. This reaction depends on both the concentration of imidazole and paranitrophenyl ester, therefore, it proceeds with a Second Order Rate Constant of 35 M-1min-1. In the second reaction, the imidazole catalyst is actually part of the substrate that is being hydrolyzed. Therefore, the rate of hydrolysis is dependent only on the substrate, and therefore proceeds with a First Order Rate Constant of 839 min-1. Rate constants of different order cannot be compared. However, the ratio of the first order rate constant to the second order rate constant gives an effective Molarity. In order for the second order reaction to be as fast as the first order reaction, it would be necessary to have imidazole at a concentration of 24 M!

Effective Concentration: 

Effective Concentration Effective concentration is k1/k2 = 2 x 105 M Effective concentration = 2 x 107 M

General Acid-Base Catalysis: 

General Acid-Base Catalysis General acid-base catalysis is involved in a majority of enzymatic reactions. General acid–base catalysis needs to be distinguished from specific acid–base catalysis. Specific acid–base catalysis means specifically, –OH or H+ accelerates the reaction. The reaction rate is dependent on pH only, and not on buffer concentration. In General acid–base catalysis, the buffer aids in stabilizing the transition state via donation or removal of a proton. Therefore, the rate of the reaction is dependent on the buffer concentration, as well as the appropriate protonation state. Specific base catalysis General base catalysis

General Base Catalysis and Ester Hydrolysis: 

General Base Catalysis and Ester Hydrolysis In the second step (collapse of the tetrahedral intermediate), the leaving group must be protonated. The general acid–base is best when its pKa is near that of the pH of the solution, in order to have appropriate concentrations of each buffer species.

Hydrolysis of Paranitrophenylacetate: 

Hydrolysis of Paranitrophenylacetate The hydrolysis of esters proceeds readily under in the presence of hydroxide. It is base catalyzed. However, the rate of hydrolysis is also dependent on imidazole buffer concentration. Imidazole can accept a proton from water in the transiton state in order to generate the better nucleophile, hydroxide. It can also re-donate the proton to the paranitrophenylacetate in order to generate a good leaving group.

Conventions for Describing General Acid/Base Catalysis: 

Conventions for Describing General Acid/Base Catalysis The dehydration reaction below is catalyzed by an enzyme at pH 7 and 25°C. This reaction does not occur nonenzymatically under these conditions. Sketch a mechanism to show how an enzyme can easily catalyze this reaction.

Dehydration Mechanism: 

Dehydration Mechanism

Electrostatic Effects: 

Electrostatic Effects Electrostatic interactions are much stronger in organic solvents than in water due to the dielectric constant of the medium. The interior of enzymes have dielectric constants that are similar to hexane or chloroform

Catalysis by Metal Ions-1: 

Catalysis by Metal Ions-1 Metal ions that are bound to the protein (prosthetic groups or cofactors) can also aid in catalysis. In this case, Zinc is acting as a Lewis acid. It coordinates to the non-bonding electrons of the carbonyl, inducing charge separation, and making the carbon more electrophilic, or more susceptible to nucleophilic attack.

Catalysis by Metal Ions-2: 

Catalysis by Metal Ions-2 Metal ions can also function to make potential nucleophiles (such as water) more nucleophilic. For example, the pka of water drops from 15.7 to 6-7 when it is coordinated to Zinc or Cobalt. The hydroxide ion is 4 orders of magnitude more nucleophilic than is water.

Covalent Catalysis: 

Covalent Catalysis There must be some advantage in any particular enzymatic reaction that proceeds via covalent catalysis. This reaction is catalyzed by pyridine, a better nucleophile than water (pKa=5.5). Hydrolysis is accelerated because of charge loss in the transition state.

Acetoacetate Decarboxylase: 

Acetoacetate Decarboxylase

Acetoacetate Decarboxylase Mechanism: 

Acetoacetate Decarboxylase Mechanism


Lysozyme Lysozyme is a small globular protein composed of 129 amino acids. It is also an enzyme which hydrolyzes polysaccharide chains, particularly those found in the peptidoglycan cell wall of bacteria. In particular, it hydrolyzes the glycosidic bond between C-1 of N-acetyl muramic acid and C-4 of N-acetyl glucosamine. It is found in many body fluids, such as tears, and is one of the body’s defenses against bacteria. The best studied lysozymes are from hen egg whites and bacteriophage T4. Although crystal structures of other proteins had been determined previously, lysozyme was the first enzyme to have its structure determined.

Lysozyme Active Site: 

Lysozyme Active Site The X-ray crystal structure of lysozyme has been determined in the presence of a non-hydrolyzable substrate analog. This analog binds tightly in the enzyme active site to form the ES complex, but ES cannot be efficiently converted to EP. It would not be possible to determine the X-ray structure in the presence of the true substrate, because it would be cleaved during crystal growth and structure determination. The active site consists of a crevice or depression that runs across the surface of the enzyme. Look at the many hydrogen bonding contacts between the substrate and enzyme active site that enables the ES complex to form. There are 6 subsites within the crevice, each of which is where hydrogen bonding contacts with the sugars are made. In site D, the conformation of the sugar is distorted in order to make the necessary hydrogen bonding contacts. This distortion raises the energy of the ground state, bringing the substrate closer to the transition state for hydrolysis.

General Acid-Base Catalysis in Cleavage by Lysozyme: 

General Acid-Base Catalysis in Cleavage by Lysozyme At what position does water attack the sugar? When the lysozyme reaction is run in the presence of H218O, 18O ends up at the C-1 hydroxyl group at site D. This suggests that water adds at that carbon in the mechanism. From the X-ray structure, it is known that the C-1 carbon is located between two carboxylate residues of the protein (Glu-35 and Asp-52). Asp-52 exists in its ionized form, while Glu-35 is protonated. Glu can act as a general acid to protonate the leaving group in the transition state. Asp can function to stabilize the positively charged intermediate. Glu then acts as a general base to deprotonate water in the transition state.

Importance of Strain in Catalysis: 

Stable Chair conformation Distorted boat conformation Importance of Strain in Catalysis

The Serine Proteases: 

The Serine Proteases The serine proteases are a class of enzymes that degrade proteins in which a serine in the active site plays an important role in catalysis. The family includes among many others, Chymotrypsin and trypsin, which we’ve talked about, and Elastase. All three enzymes are similar in structure, and they all have three important conserved residues–a histidine, an aspartate, and a serine. Chymotrypsin cleaves after mainly aromatic amino acids, while trypsin cleaves after basic amino acids. Elastase is fairly nonspecific, and cleaves after small neutral amino acids. Notice how their active sites are suited for these tasks.

Chymotrypsin Mechanism (Step 1): 

Chymotrypsin Mechanism (Step 1)

Chymotrypsin Mechanism (Step 2): 

Chymotrypsin Mechanism (Step 2)

Chymotrypsin Mechanism (Step 3): 

Chymotrypsin Mechanism (Step 3)

Chymotrypsin Mechanism (Step 4): 

Chymotrypsin Mechanism (Step 4)

Chmyotrypsin Mechanism (Step 5): 

Chmyotrypsin Mechanism (Step 5)

Chymotrypsin Mechanism (Step 6): 

Chymotrypsin Mechanism (Step 6)

Chymotrypsin Mechanism (Step 7): 

Chymotrypsin Mechanism (Step 7)

Enzyme Assays: 

Enzyme Assays In order to study enzyme reactions, there needs to be an efficient method for determining how fast products are produced by the enzyme. This is the enzyme’s activity. It is measured in µmol•min-1•mol-1 of active site (turnover number, or µmol•min-1•mg-1 of protein (specific activity). Measuring the activity of proteases is not necessarily straightforward using the normal substrates. You could for example, run a gel that might separate parent peptides from the cleaved peptides. Therefore, enzymologists make frequent use of substrate analogs that might aid in measuring enzyme activity. Serine proteases cleave ester substrates better than peptide substrates. p-nitrophenylacetate has an advantage in that the cleaved product p-nitrophenol is brightly colored yellow. The enzyme can therefore be assay in “real time.”

Burst Kinetics: 

Burst Kinetics Enzyme reactions are run under pseudo-first order kinetics. That is, the substrate concentration is so much higher than that of enzyme, that the rate of the reaction only depends on the enzyme concentration and not that of the substrate. The enzyme is considered to be “saturated” under these conditions. For all practical purposes, enzyme reactions are typically linear from T=0, until the substrate concentration decreases to below saturation level. For chymotrypsin assayed with p-nitrophenylacetate, the researchers observed a burst of p-nitrophenylacetate followed by a linear slower phase. At the same time, acetate production showed a lag followed by a linear phase having the same rate as p-nitrophenolate production.

The Rate-limiting Step: 

The Rate-limiting Step The observation of burst kinetics is suggestive of a fast step in catalysis that is followed by a slower step. The lag phase that is associated with acetate production suggests that the slow step (the rate-limiting step) is release of acetate from the enzyme active site. The rapid production of p-nitrophenolate suggests that the fast step (burst phase) is cleavage of p-nitrophenylacetate. The slow linear phase represents release of acetate from the active site. As long as it’s there, enzyme cannot bind another substrate to catalyze its cleavage. Frequently, burst kinetics is associated with formation of a covalent bond between some portion of the substrate, and an amino acid in the active site of the protein. It could also be associated with a simple slow release of products.

The Acyl Enzyme Intermediate: 

The Acyl Enzyme Intermediate Diisopropylflurophosphate is an inhibitor of chymotrypsin. It diffuses into the active, wherein a nucleophilic amino acid attacks the phosphate, releasing fluoride anion. This results in a covalent bond between the nucleophile and the inhibitor. It inhibits the reaction because it blocks entry of normal substrates. The enzyme-inhibitor adduct is very stable. Upon hydrolysis of the protein (6 N HCl, 110°C) and amino acid analysis on the hydrolysate, a novel amino acid was isolated. It was the diisopropylphosphoryl derivative of serine.

The Oxyanion Hole: 

The Oxyanion Hole The tetrahedral intermediate in chymotrypsin, which consists of the Ser195 adduct before departure of the leaving group, is considered to be the transition state intermediate in the chymotrypsin reaction. It is high energy because there is a carbon surrounded by 3 electronegative atoms, one of which bears a negative charge. How is it that the enzyme stabilizes this transition state intermediate? The backbone amides of gly193 and ser195 form an oxyanion hole. They loosely hydrogen bond to the carbonyl oxygen under attack. Upon formation of the tetrahedral intermediate, the resulting carbon-oxygen single bond is longer, and the negatively charged oxygen is better accommodated in the oxyanion hole.

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