Structural Biochemistry/Enzyme Catalytic Mechanism

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Introduction[edit]

Enzymes are proteins that can accelerate biochemical reactions. They can transfer energy forms. Their functions of catalysis are specific, and they only catalyze certain reactions and when certain substrates are present. They usually do not have side products. Enzymes only increase the rate of the reaction but not change the reaction equilibrium. In other words, with the presence of the enzyme, the reaction proceed thousands times faster, but the amount of final product is the same as without the enzyme present.

The catalytic activity usually depends on the presence of cofactors, which are not proteins. An enzyme is called apoenzyme if it is not bound to cofactors; An enzyme is called holoenzyme if it is bound to cofactors. Cofactors are divided into two groups: metal and coenzymes. Metal cofactors can catalyze in many ways. They can ease the formation of nucleophiles, stabilize intermediates, and link enzymes and substrates. Coenzymes are small organic molecules. They can be bound to enzymes tightly or loosely. They are called prosthetic groups if they are bound to enzyme tightly. There are several strategies that enzymes use to catalyze specific reactions.

1. Covalent catalysis involves a an active site with a reactive group (substrate). The active site contains a reactive nucleophilic group that attacks the substrate through covalent forces. Although the covalent interactions are temporary, the substrate is bound to the enzyme during the course of catalysis. See mechanism of the proteloytic enzyme Chymotrypsin for example of this strategy.

2. General acid-base catalysis involves acid base reactions that do not occur with water. Other molecules undergo proton accepting or donating.

3. Catalysis by approximation involves bringing two distinct substrates in close proximity which can increase the reaction rate considerably. See Nucleoside Monophosphate Kinase.

4. Metal ion catalysis involves metal ions that allow the formation of nucleophilles or electrophilles that can help the reaction occur in a faster pace.

The Nature of Enzyme Catalysis[edit]

1. Specific acid or base catalysis Enzymes are able to deprotonate or protonate a substrate by using hydrogen ion or hydroxide ion.

2. General acid or base catalysis It is similar from the specific acid or base catalysis, the reaction rate can be increased by adding or removing a proton.

3. Charge neutralization when a substrate is charged when it is bound to the enzyme, other residues of the opposite charge around the enzyme can help to maintain the binding

4. Nucleophilic catalysis Many enzymes binds substrates with covalent bonding. Enzymes always are nucleophilic. Substrates are electrophilic. Therefore, the enzymes attack the electrophilic center of substrates. This reaction is very rapid.

5. Electrophilic catalysis The enzyme reaction can be catalyzed by removing the electron.

6. Bond strain There are different types of binding in the enzyme-catalyzed reaction present such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions. When substrates bind on the active site of enzymes, their structures may not be exactly complementary to the site. These different types of binding energy contribute the binding. Further, it helps the substrates bind enzyme more tightly.

7. Environmental effects When the substrates bind the enzyme, other solvent or molecules can affect the bonding reaction. The reaction can be accelerated or deaccelerated.

Transition State Theory[edit]

The transition state theory is used to learn about relationships between a structure and the reactivity that is involved with it. To make it simpler, reagent collisions are not included in the analysis, instead the only reagents that the transition state theory deals with are the ones located at ground state. Also, the transition state theory includes the unstable components that are in the reaction. In the transition state, chemical bonds are made and they are broken. Therefore, an easy way to calculate the rate of the reaction is to assume that the reagents at the ground state are in equilibrium with the transition state, thus the concentration of the transition state is easily determined using equilibrium constants (K). Once you have the concentration of the transition state, multiply it by the composition of the reaction and you will have obtained the reaction rate. Finding the rate of the reaction is extremely important in calculating the reactivity of substrates.

To understand how enzyme working, we can start from free energy (G). The free energy difference between substrates and products is called free-energy difference (∆G). If ∆G is negative, the reaction is exergonic and it occurs spontaneously. If ∆G is positive, the reaction is endergonic and it requires additional energy to make it happen. It does nothing to the rate of th reaction, but for an enzyme catalyzed reaction, we can tell if it is spontaneous or it additional energy is needed from the value of ∆G.

In the process of an enzyme catalyzed reaction, there is a transition state before the product is formed. The difference between the substrate and the transition state is called activation energy. The use of enzyme is to lower the activation energy, so that the products can be formed more easily.

There is a region for binding in enzyme. It is called the active site. Enzymes bring the substrate into an enzyme-substrate complex. The substrates are bound to the active site. There are two models of this kind of binding. They are Lock-and-Key Model and Induced-Fit Model. In the Lock-and-Key Model, the substrate acts like a key and the enzyme acts like a lock. Since they have highly specificity, they have the perfect complementary shapes to each other. In an Induced-Fit Model, the enzyme is flexible. In other words, it can change its shape to match the shape of the substrate.

General acid-base catalysis[edit]

A general acid-base catalysis is a reaction that transfers a proton to or from another molecule. In structural biochemistry, the components that can carry a proton are substrates, cofactors, and amino acids. The reaction rate in this particular catalysis is dependent on the concentration of the proton carrier. In specific acid-base catalysis the reaction rate is independent of the concentration of the catalyst.

One example of a general acid-base catalysis is the hydrolysis of an ester. To catalyze such a reaction, the rate constant must be increased, as well as the concentration of the acid or the base by keeping a constant pH and a constant ratio of acid:base forms of the catalyst. To find out whether the catalyst is an acid or a base, increase the rate of the reaction. In the case of the hydrolysis of an ester the rate of the reaction increase is proportional to the amount of basic catalyst, therefore a base catalyst is used in this reaction. The rate of the reaction is also proportional to the catalyst’s strength. Acid-base catalysis helps stabilize the transition state. For example Ester Hydrolysis by water, the transition state is stabilized by a general base or acid. It can be stabilized by an acid group acting as a partial proton donor for carbonyl oxygen of the ester. The basic group in turn acts as a proton acceptor that stabilizes the transition state.

Example: proteases like chymotrypsin

Metal Ion Catalysis[edit]

Metal ion catalysis involves metal ions that form a nucleophilic hydroxide ion at neutral pH by activating a bound water molecule, therefore stabilizing the negative charges that are formed. At a pKa of 7, metal-bound water is extremely susceptible to nucleophilic attack. One example of a metal ion catalysis is the carbonic anhydrase mechanism in which the carbonic anhydrase catalyzes the hydration of CO2.

Another example: restriction endonucleases like BamHI and EcoRV

Covalent Catalysis[edit]

The types of enzymes that usually form covalent intermediates are serine proteases, cysteine proteases, protein kinases and phosphatase, and pyridoxal phosphate-using enzymes. An essential step in covalent catalysis is creating a covalent intermediate. A covalent intermediate encourages the reaction along to the transition state, which then in turn helps to speed-up the reaction. The formation of the covalent intermediate initiates a burst in the reaction rate followed by a decrease when the intermediate decomposes and proceeds to steady state. An example of a covalent catalysis is the Schiff base formation. The Schiff base forms by condensing a carbonyl compound with an amine.

Approximation of Reactants[edit]

There are two characteristics of enzyme-substrate binding that increase the rate of the reaction. The first is that the binding brings together substrates and the reactive groups on the active site of the enzyme. The second factor is that the enzyme active site is extremely specific in the substrates that it will bind to, therefore making the reaction as efficient as possible. Also, when the substrates bind to the active site, it causes a decrease in entropy. A decrease in entropy increases the order to the system, therefore making the reaction more favorable. The proximity effect describes the orientation and movement of the substrate molecules when binding to enzyme active sites. The orbital steering hypothesis states that just because a substrate and an enzyme active site are in close proximity does not mean that a catalysis reaction will occur. This is because the enzyme must guide or “steer” the substrate into the active site in a specific orientation in order for the reaction to actually occur. Over time, the “steering” has evolved and become more efficient.

Other requirements for such a reaction to occur is it needs a change in solvation, it needs to overcome Van der Waals forces and also changes in electronic overlap. In order for these requirements to occur, orientation effects and induction of strain are needed.

Example: NMP kinase

Isomerase[edit]

An isomerase is an enzyme that catalyses the structural rearrangement of isomer. Isomerases catalyze isomerization changes within a single molecule when isomers are equal or roughly equal in bond energy, and so they interconvert relatively freely. There are many kinds of isomerase depending on changing of structure of the molecule. For example, triose phosphate isomerase is an enzyme that catalyses an aldo sugar- Dihydroxy acetone phosphate - to the keto-sugar -Glyceraldehyde 3-phosphate by intramolecular oxidation-reduction. First, Glutamate 165 protonates the hydrogen of carbon 1 while Histidine 95 donates a proton to the oxygen atom which bonded to carbon 2. This process is forming the enediol intermediate. Later, Glutamate donated back the hydrogen, but for carbon 2 and Histidine 95 now removes hydrogen from O-H bonding with Carbon-1. This step is forming the product-Glyceraldyhyde 3-phosphate


References[edit]

Copeland, Robert A. Enzymes. Wiley-VCH, Inc., 2000. 154-70.

Fersht, Alan. Enzyme Structure and Mechanism. W.H. Freeman and Company, 1985. 47-77.

Milton H. Saier, Jr. Enzymes in Metabolic Pathways,