Enzyme Mechanism II

Last time we saw the basics of chymotrypsin, a serine protease that has three crucial amino acid residues involved in peptide bond cleavage catalysis: Ser 195, His 57, and Asp 102 - There are called the catalytic triad. The first thing we will do today is go over the mechanism of chymotrypsin in detail, and also look at the binding site in 3D.

Chymotrypsin mechanism

We had seen how the three amino acids were positioned in order to have catalysis:

Ser 195 was responsible for the nucleophilic attack on the carbonyl carbon of the peptide bond. His 57 was there acting as a base which pulled the proton off Ser 195, therefore activating it and making it a better nucleophile. Asp 102 was there for structural reasons, because it forms a h-bond to the protonated nitrogen of His 57, and locks it in position.

Now, lets go over the whole mechanism. The first step is what we have above. After this, we have the formation of the transition state. The transition state then goes into a free amine (which on the way out pulls the proton His 57 first got from Ser 195), and an acyl-enzyme intermediate, formed by Ser 195:

Now we have to complete the reaction by hydrolyzing the acyl-enzyme intermediate. For this, we get water in place of Ser 195 - Hist 57 is now deprotonated and can act as a base again. At all times, His 57 is lokcked in the right position by Asp 102:

We get a second transition state (this time is not the TS of the substrate), and when it breakes, it frees the enzyme from the acyl fragment, and Ser 195 regenerates (gets it proton back) from His 57:

The products are released, and the enzyme is regenerated. Note that the kinetic mechanism of this enzyme corresponds to a ping-pong mechanism, where the substrates are the peptide to be cleaved and water.

X-Ray structure

Unofrtunately, the only way to see this is with CHIME. Here are models of trypsin (another serine protease) without and with their substrate bound. Try to find His 57, Ser 195, and Asp 102. Are they on the surface of the protein? Are they inside a cleft formed by different subunits of the protein? How are things placed with respect to each other?

Acetoacetate decarboxylase

Now we will study the mechanism of an enzyme that we saw briefly before. Acetoacetate decarboxylase catalyses the decarboxylation of b-keto acids (that is, molecules in which there is an carboxylic acid two positions away from a ketone). The non-enzymatic mechanism of this reactions is:

Several studies have shown that a lysine residue is required for good activity of the enzyme. How can a Lys catalyze this reaction? If you remember, an amine can activate a carbonyl residue by forming a Schiff base, which is sort of a black hole for electrons. Now, the first step of the reaction is the formation of a Schiff base. This step is catalyzed either by general or specific acids and bases.
 
 

Now that we have the Schiff base in place, the carbon b to the carboxyl became a lot more electropositive because we have an electron sink on the carbon - The decarboxylation proceeds really fast:

We see that there is no charged intermediate using a Schiff base. Actually, we lose charges if we decarboxylate. After we lose CO₂, we have reformation of the Schiff base, and water then hydrolyses (again using general acid-base catalysis) the Schiff base to the final product and the free enzyme:

Aldolases

So far we studied enzymes that break stuff. Actually, we studied the 'breaking' direction of the reaction. We have to remember that an enzyme catalyzes both the forward and the reverse reaction. We will now describe an enzyme that actuallty makes something, aldolase. Aldolase catalyses a reaction that probably rings a bell from organic chemistry, the Aldol reaction. It is used heavily in the synthesis of sugars from smaller fragments, usually 3-carbon fragments.

As acetoacetate decarboxylase, aldolase uses a amino group from a lysine residue to form a Schiff base, and make a carbonyl group even more electrophilic (make it an electron sink). In the first leg of the reaction, the first half of the final sugar, which is phosphorilated, reacts with the amine base to form the Schiff base:

After the electron sink is formed, one of the carbons of the 3-carbon becomes very nucleophilic (the one a to the imine). This one attacks the aldehyde portion of the second fragment of the sugar:

In the final steps, we have hydrolysis of the Schiff base, regeneration of the amine base (Lys), and release of the sugar-phosphate:

Another important group that forms Schiff bases is pyridoxal phosphate (vitamin B6). This group can be found as an aldehyde (pyridoxal phosphate) or as an amine (pyridoxamine phosphate). It will therefore be able to form Schiff bases either with amines or with carbonyls:

As an aldehyde (reacts with amines):

As an amine (reacts with carbonlys):

This group can help in a number of enzymatic reactions, including decarboxylations, transaminations, and racemizations.

Enzyme regulation

So far we have studied how enzymes work, but not when they work. In the organism, the action of certain enzymes (proteases for example) has to be carefully regulated. If a protease worked whenever it wanted to, it would chew up on proteins and, for example, go through muscle like butter. Not a pretty picture. The same goes for other enzymes that catalyze other reactions.

Nature has designed enzymes so that they can be regulated.  cases where the enzyme is part of a larger metabolic pathway, products from enzymes downstream in the pathway can act as inhibitors of the first enzyme in the pathway. One nice example is the conversion of L-threonine to L-leucine. There are five enzymes in charge of this. The first enzyme, L-threonine dehydratase, is inhibited by the end product (L-leucine). This is called feedback inhibition:

There are different ways by which the activity of an enzyme can be controlled.

- We saw a way of controlling protein binding when discussing hemoglobin. An enzyme can use allosteric control to do feedback inhibition. L-threonine dehydrogenase (LTDH) is an allosteric enzyme (above), which has a binding (not active) site for L-leucine. After L-leucine reaches high levels in the system, it will start binding to LTDH. Its conformation will change, and the forward reaction is slowed down. This will in turn make the other enzymes in the pathway slow down, and therefore the system has time to deal with all the L-leucine it made before making more.

All the things we learned for hemoglobin apply here. In some cases, the activity of the enzyme depends on the concentration of substrate. That means, the activity of the enzyme is modulated by the substratre - They shown homotropic modulation (cooperativity). In this cases, the cooperativity is almost always positive.

In other cases, the modulator is different from the enzyme (LTDH), and we have negative cooperativity. One thing that we have to remember, is that instead of having an hyperbolic Vmax vs. [S], the curve will be a sigmoid - The enzyme does not follow Michaelis-Menten kinetics. K_M is therefore not K_M, but K_0.5 (it still represents the concentration of suibstrate at which the rate of the enzyme is half of V_max).

- Another way of achieving feedback inhibition is achieved by covalent modification of the enzyme. Either the covalent modification is needed to make the enzyme active, or the covalently modified enzyme is inactive. In the same way that allosteric control, the covalent modification can cause a change in conformation that activates or deactivates the enzyme.

Covalent modification can also inutilize an amino acid residue that is required for catalytic activity. In either case, the adition and cleavage of the molecule modifying the enzyme is in charge of another enzyme, which is a regulatory enzyme.

Phosphorilation is one of the best known ways enzymes have to be triggered on and off. The enzyme glycogen phosphorylase (GP) is regulated in this fashipon. This enzyme catalyses the convertion of glycogen (which is a polysaccharide of glucose) into smaller chunks of glycogen and glucose-1-phosphate:

(glucose)_n + P_i <> (glucose)_n-1 + glucose-1-phosphate

The glucose-1-phosphate is then broken down to glucose in the liver, or used up in the muscle. GP exists as two forms, a and b. GP-a is a dimer which has two phosphorilated serine residues, Ser-O-PO3^-, and is the active form of GP. A second enzyme, phosphorylase kinase (PK), is in charge of regulation. PK takes GP-b and converst it into GP-a using 2 molecules of ATP (denosine triphosphate):

GP-b + 2ATP <> GP-a + 2ADP

What regulates the activity of the enzyme in this case is a conformational change in the shape of GP upon phosphrilation:

Another way of controling the way enzymes work is to have them as an inactive form until they are required. This is usually found with proteases. The enzymes are first made as inactive precursors, called zymogens. Zymogens are either longer polypeptide chains which have extra amino acids that render them inoperative, or they have subunits that need to be separated tied together with intersubunit peptide bonds.

After they are where they are suposed to, other regulatory enzymes chop the unwanted polypeptide chains out of the way, or cleave the different subunits free. A good example is chymotrypsin. The enzyme is synthtized as chymotrypsinogen, which is a single chain. Trypsin first cuts between residues 15 and 16 to give p-chymotrypsin, which is active. p-chymotrypsin then catalyzes its own conversion into a-chymotrypsin:

In these cases, activation is ireeversible. The body synthetizes protease inhibitors (usually peptides) of its own to shut off the activity of the proteases.

Next time we will hopefully start with lipids and membranes.

Prepared by Guillermo Moyna, 1999.