Functional Principles of Bionanotechnology-3.pptx

Functional Principles of Bionanotechnology-3

1. Chemical Transformation

2. Chemists have been performing specific chemical transformations for centuries, creating structurally pure molecules for use in medicine and industry. This process typically proceeds through a sequence of specific chemical modifications, such as adding a new group, changing a specific bond, or making other small changes. At each step, a new reaction must be designed, often with significant side products competing with the desired reaction. The chemist must purify the desired product at each step and search for reactions that maximize the proper products. For an organic molecule with dozens of atoms, like many of the molecules used as drugs, this may involve dozens of steps and the final yield may be quite low.

3. Biological machinery excels in one ability above all others: performing specific chemical transformations. Like chemists, cells create specific molecules by an ordered set of small chemical transformations. But, unlike the chemist, they build specific bionanomachines —enzymes—that perform each step efficiently and accurately (Figure). Typically, each enzyme is optimized to perform a single reaction, speeding the chemical process by a trillionfold or more over the unassisted rate. The formation of unwanted side products is reduced nearly to zero, so that the desired product, even if it requires dozens of synthetic steps, is created at high yield (Figure). These machines are also carefully regulated, so that products may be created when and where they are needed. Enzymes are a priceless gift from nature, providing the starting point for all of bionanotechnology.

4. Figure Triose phosphate isomerase is an example of a perfect enzyme. It is a diffusion-limited enzyme, performing its reaction at rates faster than the rate at which substrate molecules can diffuse to it. It uses all the tricks employed by enzymes to perform their reactions. The enzyme is a dimer of two identical subunits, each with a separate active site that surrounds the substrate molecule, shown in pink. Note how large the enzyme is relative to the substrate. All this infrastructure is needed to ensure that a handful of key active site amino acids are perfectly arranged.

5. Figure Triose phosphate isomerase performs an isomerization reaction, removing two hydrogen atoms (shown in red) from dihydroxyacetone phosphate and replacing them in different positions to form glyceraldehyde 3-phosphate. The reaction is performed in two steps, using two key amino acids. In the first step, shown at the top, a glutamate extracts one hydrogen atom and a histidine adds another (shown in dark red) back to the molecule in a different position. Then, in the second step, the glutamate replaces its hydrogen atom in a different position and the histidine grabs another hydrogen atom from the substrate. Note that the enzyme starts and ends in the same form—with a free glutamate and with hydrogen bound to the histidine (albeit a different hydrogen atom). This leaves the enzyme ready to perform the same reaction on the next substrate molecule that it encounters.

6. The phosphate group of the substrate is surrounded by a collection of amino groups from the protein, shown here in dark gray on the left. They form a specificity pocket that forms hydrogen bonds with the substrate, positioning it correctly relative to the catalytic histidine and glutamate amino acids above. The enzyme speeds the reaction by stabilizing the transition states of the two steps. The transition state of the first reaction is shown here on the right. The glutamate has extracted one hydrogen, but the histidine has not yet donated its hydrogen. This leaves an unfavorable negative charge on one oxygen atom in the substrate. This is stabilized by a lysine amino acid from the enzyme, which carries a positive charge.

7. Enzymes perform chemical transformations by paving the way through the desired reaction, smoothing over any obstructing hills and lowering any roadblocks. A chemical reaction is similar to the process of breaking a pencil. At the beginning the pencil is perfectly solid and static. Then you start to apply pressure and it bends, resisting and straining all the way. Finally, it snaps, and you are left with two pieces, solid and static. A chemical cleavage reaction is similar. If you are breaking a molecule in two, you must pull it forcibly apart. The beginning and final forms—the molecule and the two halves—are perfectly stable, but the intermediate states, as individual bonds are stretched, are highly unstable and energetically unfavorable.

8. Figure Cells and synthetic chemists both build organic molecules in a series of chemical transformations, taking available starting materials and making chemical changes until the desired product is obtained. The refinement added by natural systems is the use of enzymes at each step that provide specificity and efficiency that are not available in the solution processes typically used in organic chemistry. This is the sequence of steps used in bacteria to build penicillin from the common small molecule pyruvate. Two pyruvate molecules are combined and converted into the amino acid valine in four steps. Then it is combined with two other amino acids, each also created by a number of steps from simple precursors, to form the basic skeleton of penicillin. Three additional steps create the active form.

9. Enzymes reduce the energetic barrier imposed by these intermediate states—termed transition states—making them easier to form from the starting material and easier to convert into the proper products. Think again of the pencil. This time, take your fingernail and make some deep dents in the wood along the side. Now, the pencil bends and breaks far more easily. You have catalyzed the pencil-breaking reaction by changing the intermediate states with your fingernail, making the partially bent states easier to achieve. Similarly, enzymes create a molecular environment in which the transition states are stabilized so that they do not present such a barrier to the reaction.

10. All of the action occurs in the active site of an enzyme. There, specific amino acids are placed in strategic locations, perfectly positioned to stabilize the transition state of the molecule undergoing the reaction. Many diverse methods are used, each tailored for a given reaction. At first glance, every active site seems to be different, each developed separately by evolution for its task. But several general principles are used in most cases: reduction of entropy, chemical stabilization of the transition state, and use of specialized chemical tools. The three-dimensional structures of hundreds of enzymes have been solved and are available through the Protein Data Bank (http://www.pdb.org). This is a primary resource for bionanotechnology, providing a wealth of working examples of specific chemical catalysts. These provide an excellent starting point for the development of custom enzymes, tailored for nonbiological applications.

11. Enzymes Reduce the Entropy of a Chemical Reaction Enzymes speed reactions by having everything at the right place at the right time. Entropy is constantly diluting reactions, reducing the probability that molecules will meet and react in the desired way. In a reaction that requires the joining of two molecules, entropy will ensure that they rarely find one another. In a reaction that requires an exact alignment of two bonds, entropy will ensure that this alignment is just one among many other random alignments. Enzymes are nanoscale jigs that fight entropy by positioning reacting molecules and forcibly aligning reacting bonds in the proper orientation.

12. Active sites conform closely to the shape of the molecules being transformed. The surface of the active site is complementary to the molecule. It will have carbon-rich patches abutting carbon atoms in the molecule and hydrogen-bonding atoms in perfect registration with hydrogen-bonding atoms on the molecule. Enzymes commonly make contact with most of the molecule, and some enzymes completely enclose their targets, using flaps and doors that close after the molecule is bound. The active site is typically separated into two functional regions. First, there is a specificity pocket that recognizes the proper substrate and binds tightly to it. Second, there is the catalytic machinery that performs the chemical transformation.

13. A separate specificity pocket, often comprising most of the active site, is needed in most cases because the catalytic machinery is often chemically exotic and must be optimized to perform the chemical reaction instead of providing the optimal binding characteristics. The typical tolerances for complementarity between enzymes and their substrates are very fine, fitting together to a fraction of a nanometer. These high tolerances make enzymes highly specific in the reactions that they catalyze. Natural enzymes routinely separate molecules that differ by a single atom. They also can be highly stereospecific, separating right-handed and left-handed forms of a molecule or creating only one of many possible forms.

14. Enzymes Create Environments That Stabilize Transition States After substrates are locked comfortably into the form-fitting, reduced-entropy active site, enzymes create a chemical environment that promotes the desired chemical transformation. This is the heart of nanotechnology, where specific atoms are removed or added according to demand. Enzymes catalyze reactions by stabilizing the intermediate, transition state of the reaction. This is accomplished in many ways, tailored to the given reaction. In some cases, chemical groups, provided by surrounding amino acids or prosthetic groups, interact with the substrate, modifying its electronic structure to make portions more reactive. A charged amino acid can polarize a neighboring bond in the substrate, making a target atom more susceptible to attack or making it a stronger attacker. A hydrogen bond may stabilize a form of the substrate that is normally found with low probability but is the right shape for the desired reaction.

15. In many cases, the transition state may include an unstable charged form of the molecule, with one or more atoms in a less than ideal bonding state. These are often stabilized by placing an amino acid nearby that carries the opposite electronic charge, forming a stable electrostatic interaction with the transition state. Enzymes can also introduce geometric strain in substrates. In cases in which the geometry of the substrate changes during the reaction, the active site is designed to fit more tightly to the shape of the transition state than to the initial substrates, favoring the transformation from substrate to transition state.

16. Enzymes Use Chemical Tools to Perform a Reaction Most enzymes use specific chemical tools to interact directly with the substrate, directly making chemical changes. It is important that these tools end up, after the reaction is performed, in the same state that they begin. This is the definition of a catalyst, which may undergo a chemical change to assist the reaction, but which must be restored at the end so that it is ready to perform subsequent reactions. Enzymes commonly use reactive amino acids to make specific chemical changes in substrates. For instance, many enzymes use key amino acids to shuffle hydrogen atoms within a molecule. In particular, histidine is often used in this role. At the pH of a typical cell, it is fairly easy to remove one of the hydrogen atoms on histidine amino acids and later replace it. Histidine is used in many reactions that remodel molecules. A histidine in the enzyme will pull a hydrogen atom off the molecule and then replace it in a slightly different position, flipping the handedness of the molecule or moving the location of a double bond.

17. Other reactions require a more forceful approach. In these enzymes, an amino acid attacks the substrate, forming a covalent bond. Typically, the bond is quite unstable, and a subsequent step will break the bond, forming the desired product. Serine proteases are one example, where a serine amino acid attacks a peptide substrate, breaking the chain and forming an unstable bond to one half. Soon after, a water molecule enters and separates the remaining half, restoring the serine to its original form.

18. In some reactions, enzymes add new atoms to a growing molecule. When you are going to build a new deck at your house, you rarely start from raw materials. You don’t chop down trees and mine iron ore; rather, you go to the lumberyard and buy two-by-fours and nails. Similarly, enzymes often build new molecules with prepackaged atoms that are easy to add to a growing product. Many molecules serve as carriers for specific atoms (Figure ). ATP carries a phosphate group, and other molecules are carriers for carbon atoms, sulfur atoms, nitrogen atoms, and hydrogen atoms. These molecules are cleverly designed: All hold their atoms with an unstable bond, so they are easily released to their new position. These coenzymes are often exotic-looking molecules that must be synthesized specially for the job.

19. Some reactions simply require the shuffling of a few electrons. Metal ions typically play the role of electron carriers, because they can cycle between several stable charged forms. Copper and zinc ions are often held tightly by a small cluster of amino acids. Iron, on the other hand, is often trapped in the middle of a large heme molecule, which is held in turn within a tight pocket in the enzyme. Unusual metals, such as molybdenum and vanadium, are used when real force must be applied, as in nitrogenase, the enzyme that separates the two tightly bonded atoms in nitrogen gas.

20. Specialized prosthetic groups are used to deliver raw materials to synthetic enzymes. The three shown here are each composed of three parts. At the left in gray is an adenine group, which is used as a handle for holding the prosthetic group in place. This is attached to a specially designed chemical group, shown here in pink, that loosely holds the raw material, shown in red. The phosphate in ATP is easily removed because it is chained uncomfortably close to two neighboring phosphates that carry negative charges. NAD carries a hydride ion (a hydrogen atom with an extra electron). When released, the ring switches to a more stable aromatic structure. Adenosylmethionine ( adoMet ) carries a methyl group that is held on a sulfur atom. When the methyl group is removed, the sulfur returns to its preferred state, bonded to two atoms instead of three.