enzymes and reaction mechanisms and serine proteases

how do enzymes change the activation energy of a reaciton

Enzymes lower the activation energy of the reaction. Enzymes do this by binding to the reactant molecules and holding them in such a way as to make the chemical bond-breaking and forming processes take place more easily. It is important to remember that enzymes do not change whether a reaction is exergonic (or spontaneous) or endergonic. This is because they do not change the free energy of the reactants or products. They only reduce the activation energy required for the reaction to go forward. An enzyme itself is unchanged by the reaction it catalyzes. Once one reaction has been catalyzed, the enzyme is able to participate in other reactions.​

Activation energy conditions consider bond rearrangements, alignment of reacting groups, and formation of transient unstable charges among others.​

impact of an active site on an enzyme?

All enzymes are proteins except for a few RNA catalysts. Enzymes need to be in their native state to catalyze the reaction, so they can’t be dissociated into subunits or denatured (unfolded). If they are broken into residues, their catalytic activity is destroyed. They must have the correct structure, from primary to the highest structure to be active. The active site is a pocket where the reaction takes place and the reactant is the substrate. It’s the molecule that the enzyme acts upon. The active site is lined with residues that bind with the substrate. Here arginine residues help orient the substrate by the interactions with the substrate’s phosphate group. The reaction is catalyzed in the pocket which is removed from the aqueous environment of the cell. Enzymes are specific and can discriminate between substrates, even if they are similar structurally. Each enzyme usually only catalyzes one reaction

impact of cofactors

Cofactors provide additional chemical groups to help facilitate enzymatic reactions. Metal cofactors such as zinc help bind the substrate and iron is a cofactor in oxidation reduction catalysis. Not having trace elements in the diet interferes with catalysis and shows up as pathology such as iron deficiency anemia. Heavy metals like lead can take the place of cofactors and block enzyme function.​

apoenzyme

protein portion, inactive

holoenzyme

whole enzyme, active

oxidoreductases

The first class to look at is the oxidoreductases and these catalyze redox reactions. Redox reactions are reduction and oxidation by transferring hydrogen or oxygen atoms.

transferases

Transferases we’ve seen before, most notably kinases where we have transferred a phosphoryl group and have moved the phosphate. Transferases also act in methylation or the movement of other small groups.

hydrolase

Hydrolase forms products by hydrolyzing a substrate.

lyases

Lyases cleave carbon and other bonds by catalyzing addition or elimination reactions and can form new ring structures or double bonds.

isomerases

Isomerases rearrange groups within a molecule to form isomers.

ligases

Ligases are synthetases that use ATP to put molecules together.​

what are active sites complementary to

Active sites are complementary, not to the substrate, but to the enzyme transition state. Here in B, we see that the active site is lined by magnets in this example. They bind tightly in the active site so the activation energy would be large because it is not favorable to bend the stick and this inhibits products from forming. The delta G-m for magnetic is the same as the delta G-b for binding and the delta Gb energy is released when bonds are formed in the active site. Enzymes are complementary to the transition state not the substrate.​

acid base catalysis

Acid-base catalysis is one of the three most common catalytic strategies in enzyme active sites. Many reactions pass through unstable, charged intermediates, and these are stabilized by moving protons. When water carries out the proton transfer, the process is called specific acid-base catalysis. When a functional group on the enzyme carries it out, the process is called general acid-base catalysis. A general acid donates a proton to the substrate, and a general base removes a proton from the substrate.​

Several amino acid side chains can do this, including histidine, aspartate, glutamate, serine, threonine, tyrosine, cysteine, lysine, and arginine. The acid form of each side chain is the proton donor, and the base form is the proton acceptor. Whether a side chain acts as an acid or a base depends on its pKa. The microenvironment of the active site shifts the pKa of a residue away from its value in free solution, which places the residue in the right protonation state at the right time. Histidine is the best example. Its side chain has a pKa near 7, so it exists in both the protonated and deprotonated forms at physiologic pH. This lets histidine serve as either a proton donor or a proton acceptor.​

covalent catalysis

Covalent bonds are transient but stabilize acyl intermediates and lower activation energy to have the reaction proceed. The chemical group in the active site acts as a nucleophile. The hydroxyl is used to form a bond between the enzyme and the substrate. Hydrogen is removed in bond formation and then water is used to release the product from the active site.​

In covalent catalysis the active site R group is usually a nucleophile. The electron-rich oxygen atom attacks carbon, and nitrogen and sulfur also act as nucleophiles.​

metal ion catalysis

Metal ions can shift electron density so that a bound water molecule loses a proton and becomes a nucleophile. Three His residues in the active site of carbonic anhydrase coordinate a Zn ion. Zinc binds water and reduces its pKa from 15 to 7. In the process, water loses a proton, carbon dioxide is able to react with the hydroxide ion for the formation of bicarbonate ion. The product is released from the active site and the enzyme is regenerated.​

common structural components of serine proteases

For serine proteases, the most conserved amino acids are found in the active sites. The binding pocket binds to specific amino acid residues within the protein substrate. What binds in the binding pocket (yellow) is aligned and properly oriented so that the catalytic amino acids in the active site (red) can break peptide bonds adjacent to the amino acid sitting in the binding pocket. The binding pocket is also considered the specificity pocket because it determines which peptide bonds are going to be broken and so determines the specificity of the particular serine protease.​

Amino acids in red are the catalytic triad. They are a set of three amino acids within the active site that are conserved in all serine proteases and are essential for the activity of the serine protease. The oxyanion hole stabilizes intermediates.​

Here is a close-up of the active site with a substrate (white and yellow) bound. The hydroxyl of Ser195 attacks the carbonyl group of the substrate (the oxygens are red); the developing negative charge on the oxygen is stabilized by the oxyanion hole (amide nitrogens from Ser195 and Gly193, in blue). The aromatic amino acid side chain of the substrate (yellow) sits in the hydrophobic pocket. The amide nitrogen of the peptide bond to be cleaved (protruding toward you and projecting the path of the rest of the substrate polypeptide chain) is shown in white.​

substrate specificity of serine proteases

Each serine protease binding pocket accommodates different amino acid residues and different peptide bonds are oriented in the active site for cleavage. Chymotrypsin has a deep binding pocket that is also wide and primarily hydrophobic. It cleaves peptide bonds after bulky aromatic R groups like phenylalanine, tryptophan and tyrosine. The pocket for trypsin is more narrow so won’t accommodate big rings. Trypsin has a negative charge at the bottom that attracts positive charges on positively charged side chains so cleaves peptide bonds after residues like arginine and lysine. Elastase has a shallow pocket and only accommodates small residues like alanine so it cleaves next to alanine and glycine residues.​

the catalytic triad

The three catalytic amino acids are a serine, histidine and aspartate. Numbers vary depending on the serine protease, but the order and arrangement are the same. Aspartate and histidine work together to make serine a nucleophile so it can attack the peptide bond.​

role of the oxyanion hole

The oxyanion hole is formed from the backbone amino groups of two residues. So here in chymotrypsin it’s Glycine 193 and Serine 195. This serine is also in the catalytic triad. The hole is where a charged oxygen can sit and interact via hydrogen bonds. During the reaction of chymotrypsin, two short-lived and highly unstable tetrahedral intermediates are formed that contain an oxygen with a formal negative charge. This intermediate is representative of the transition state, which is a high energy state that the reactant must pass through on its way to product. The negative charges form hydrogen bonds with the hydrogens in the oxyanion hole and that stabilizes the high energy intermediate. So these extra bonds have binding energy in catalysis.​

chymotrypsin mechanism step 1: substrate binding

The chymotrypsin mechanism occurs in 6 steps. In diagram 1 we see an empty active site with the substrate specificity pocket again shown in yellow. We can also see the catalytic triad residues of Aspartate 102, Histidine 57 and Serine 195. The oxyanion hole is shown in orange with the backbone amino groups of Glycine 193 and Serine 195 on either side. So step 1 of the mechanism involves the peptide substrate binding to the enzyme active site. Phenylalanine fits into the substrate specificity pocket and the enzyme cleaves the bond adjacent to that R group. The next residue on the polypeptide is not specific to the reaction.​

Chymotrypsin will cleave the peptide bond between the amino acid residues and separate the green amino acid from the blue amino acid.​

In diagram 2, the substrate binds to the active site and the aromatic R group of phenylalanine binds to the specificity pocket which positions the carbonyl oxygen of the peptide backbone in the oxyanion hole and it positions the peptide bond between the carbonyl and the amino group adjacent to the serine in the active site. This is the bond to be broken.​

second step of chymotrypsin mechanism

The binding of the substrate causes a subtle conformational change that shortens the hydrogen bond between Asp102 and His57 (arrow 1). His57 then removes the proton from Ser195 (arrow 2), which allows the serine oxygen to attack the carbonyl carbon of the peptide (arrow 3). This forms a covalent bond between the enzyme and the substrate. The attack pushes a pair of electrons from the carbonyl double bond onto the oxygen, producing an oxyanion, which is a negatively charged oxygen (arrow 4). The oxyanion hole stabilizes it through hydrogen bonds with the backbone NH groups of Gly193 and Ser195. The result is the first tetrahedral intermediate, shown in the right panel. The central carbon now has four attachments in a tetrahedral arrangement, which resembles the transition state. The extra interactions between this intermediate and the groups in the active site stabilize it and lower the activation energy. The acyl-enzyme intermediate forms in the next step, when this tetrahedral intermediate collapses in step 3.​

third step of chymotrypsin mechanism

In step 3, the pair of electrons on the oxygen are going to return to the double bond (arrow 1 on both panels) and so the tetrahedral intermediate resolves itself. The electrons shared in the bond are taken by the nitrogen (arrow 2) and a proton is donated by His 57 (arrow 3) so it acts as a proton donor. The formation of new bonds causes a slight conformational change that then lengthens the hydrogen bond between Asp 102 and His 57 returning the pKa of His57 to its normal value so that it can donate a proton again. Breaking the peptide bond releases the carboxyl-terminal fragment of the substrate from the active site. This fragment leaves with a free amino group at its new amino terminus. The amino-terminal fragment stays behind, covalently bonded to the serine oxygen of the catalytic triad through its backbone carbonyl carbon. This covalent complex is the acyl-enzyme intermediate.​

step 4 of chymotrypsin mechanism - water entry

To resolve the acyl enzyme intermediate, a water molecule enters the active site. His 57 can act as a base and remove a proton from water so that the hydroxyl that forms can attack the carbonyl carbon of the backbone (arrow 1) and that attaches the hydroxyl group from the water to the carbon and forms a second tetrahedral intermediate. The carbon has 4 attachments now with the hydroxyl group and the oxyanion is stabilized once again in the oxyanion hole.​

final steps of chymotrypsin mechanism

The oxyanion resolves itself, and the electrons from the oxygen return to the double bond (arrow 1), which breaks the bond between the carbonyl carbon of the substrate and the serine oxygen. This releases the amino terminal fragment of the substrate from the enzyme. His57 acts as a general acid and donates a proton to the serine oxygen (arrow 2), which regenerates Ser195 and resets the active site so that it can once again participate in a hydrogen bonding network as seen again in diagram 6.​

This is quite a lot! There are 4 main points to know about this mechanism:​

1. The positioning of the catalytic amino acid triad in the active site creates the nucleophile that can cleave the peptide bond.​

2. The nucleophilic attack by serine creates a transient covalent acyl-enzyme intermediate, which is an example of covalent catalysis.​

3. The positioning of glycine and serine create a micro environment called the oxyanion hole which stabilizes an otherwise unstable tetrahedral intermediate.​

4. Histidine serves as a general acid base catalyst throughout the mechanism, withdrawing a proton to increase the nucleophilicity of serine and donating a proton to resolve the tetrahedral intermediates.​