Where do molecules obtain the needed activation energy? From the molecular storm! The impetus needed to make it across the transition state is provided by small, fast molecules (typically water molecules) fortuitously colliding with the reacting molecules to give them the right push. If lucky, the push causes the molecules to snap into their new shapes. Of course, not every colliding water molecule will have enough energy or hit the reacting molecules in the right way. Chemical reactions take time— we have to wait for the right push to come along, and the higher the activation energy needed, the more time it takes, as higher-energy collisions are much less frequent than low-energy collisions.
What determines the height of the activation barrier? In a nutshell, it depends on how uncomfortable the molecules become as they transform from the initial to the final state. If I try to change a molecule by rearranging several of its bonds, it will take a lot of bending and twisting, and activation energy will be high. On the other hand, if a tiny rearrangement of one atom will do, activation energy may be low.
Many important chemical reactions in cells require enormously large activation energy. These transformations proceed at such a glacial pace we’d probably be dead before our cells cycled through them even once. To solve this problem, nature has invented a neat trick. Let’s imagine a helper molecule whose job is to make the transition state more comfortable and therefore lower the activation energy (Figure 6.3). If the activation energy is lowered, the reaction rate increases. Such helpful molecules are called enzymes, and their involvement speeds up reactions by many orders of magnitude. Enzymes are biological catalysts, comparable to your car’s catalytic converter, which speeds up the oxidation of harmful exhaust gases.
FIGURE 6.2. Chemical reactions proceed via “uncomfortable” transition states.
Enzymes have a pocket that is custom-made to fit the substrate (the reactant molecules). Initially, when the substrate binds into the enzyme’s pocket, it is not a perfect fit, but as the enzyme and the substrate slightly change shape and begin to fit more comfortably, the enzyme pushes the substrate toward the transition state. Once the substrate is in the transition state, the enzyme is happy (at lower energy), and the total energy of the system (the energy of the transition state plus the energy of the enzyme) is reduced (Figure 6.3). Because the transition state still has higher energy than the final, product state, the transition state has no choice but to convert to the product. The product is uncomfortable in the enzyme’s pocket and is released. The enzyme snaps back to its old shape and is ready to take on a new substrate for conversion.
FIGURE 6.3. How an enzyme accommodates a transition state and therefore lowers the activation energy of a chemical reaction.
To illustrate the remarkable abilities of enzymes as catalysts, consider an enzyme called phosphoglucomutase. This enzyme converts an indigestible kind of sugar into a more palatable one. By speeding up the conversion process by a factor of one trillion, one phosphoglucomutase molecule can convert a hundred sugar molecules per second. Without this enzyme, it would take three hundred years to convert just one sugar molecule!
Enzymes are large molecules made of proteins—long chains of amino acids that fold into complicated shapes, as described in Chapter 4. Proteins are like a box of Legos: By changing the sequence of amino acids, almost any shape of protein can be constructed. Using the trial and error of evolution, myriads of enzymes have evolved, each custom-made to catalyze a particular reaction.
Molecular machines can be considered a special type of enzyme. As will be described later, they have evolved from enzymes. Enzymes and molecular machines share many attributes. In some sense, enzymes are machines: They bind, transform, and release molecules by virtue of their specific structure, but also as a result of being jostled by the molecular storm. A molecule does not know how it will fit into the binding pocket of the enzyme. Instead, the constant bombardment by water molecules (about every 10−13 seconds) rapidly rotates and deforms the molecule. After a millisecond, by chance, the molecule is pounded into the right shape and orientation to form a complex with the enzyme. If you are amazed that the molecule is able to find the right orientation and shape, consider that in a millisecond, an average molecule undergoes ten billion collisions with water molecules.
Being in Control
Enzymes are amazing molecules, but they are not what we would call alive. First of all, they do not move. They deform a little bit when binding a substrate molecule, but this seems hardly sufficient to operate a molecular machine.
In the early days of biochemistry, biologists envisioned the cell as a pouch containing enzymes that catalyzed various reactions. The cell was thought of as nothing more than a tiny reaction vessel. It soon became clear, however, that cells would not survive long if enzymes were given free rein to make and break down molecules. Cells have to respond to their environment. Cells have to be able to make decisions.
Computers can make decisions because computer programs contain logical commands such as “IF . . . THEN. . . .” In a cell, logical decisions are implemented on a molecular scale. Part of this cellular software is, of course, DNA, which contains the blueprint for how to make different proteins and how to guide a body through its development. But DNA does not control the day-to-day operation of the cell. This is done by enzymes and molecular machines. So the real question is, how do enzymes compute?
This mystery was solved in the early 1960s by Jean-Pierre Changeux (b. 1936), a young graduate student in Jacques Monod’s laboratory at the Pasteur Institute in Paris. Studying an enzyme called L-threonine deaminase, which broke down its substrate, the amino acid threonine, he found that the enzyme’s activity was inhibited in the presence of the molecule isoleucine, another amino acid. Inhibition of enzymes had been seen before and was thought to occur when the enzyme was bound to a molecule other than its substrate. This process of an unwanted houseguest’s clogging up the binding pocket of an enzyme is called competitive inhibition. A familiar example is carbon monoxide poisoning: This gas will fit so strongly into the oxygen-binding pocket of hemoglobin (the oxygen-carrying proteins in our red blood cells) that it cannot be released again. In the competition between oxygen and carbon monoxide, carbon monoxide is the clear winner. Once the hemoglobin in a victim’s blood becomes gummed up with carbon monoxide, oxygen transport is shut down, and the hapless victim suffocates.
Changeux’s deaminase did not follow the model of competitive inhibition, however. When he tried to shut down the isoleucine binding site on the enzyme, the enzyme continued its enzymatic activity uninhibited. If the isoleucine binding site and the catalytic binding pocket had been one and the same—as in the case of hemoglobin—the enzyme’s ability to bind the threonine substrate should have been impeded. Changeux concluded that the enzyme had two binding pockets: one for threonine (the enzyme’s substrate), and one for the isoleucine, which regulated the enzyme’s activity.
In his autobiographical paper “Allosteric Receptors: From Electric Organ to Cognition,” Changeux recalls that he first presented his startling findings at the Cold Spring Harbor Symposium on Quantitative Biology in 1961. Here, he told his fellow scientists that “the interaction between these two sites was indirect and transmitted by a conformational change of the protein molecule.” In other words, binding the isoleucine caused a large change in the shape of the enzyme, closing down the binding pocket for the substrate, and rendering the enzyme unable to process threonine molecules (Figure 6.4). The isoleucine acted as a control molecule—it was as if in a computer program, there were a line: “IF isoleucine is present THEN do not process threonine.”
FIGURE 6.4. Top: An allosteric enzyme combines two smaller molecules (its substrate) into one larger molecule by binding them to a suitable pocket in its structure (active site). A control molecule (right) approaches the enzyme. Bottom: The control molecule has attached itself to the enzyme’s control site. This has caused the enzyme to change shape (conformation) and close off the active site. The enzyme halts its cat
alytic activity as long as the control molecule remains bound.
Much enzymatic activity in living cells is controlled by the binding and release of such control molecules. Binding a control molecule changes an enzyme’s shape, with part of the enzyme moving relative to other parts. This change in shape can increase or decrease the enzyme’s catalyzing activity by opening or closing the pocket that binds the enzyme’s substrate. The control molecules can also completely shut down the enzyme. This ability of a control molecule to change the enzyme’s shape and activity was named allostery by Changeux’s thesis advisors, Jacques Monod and François Jacob.
Once you have allostery, you can implement many useful schemes to control chemical reactions in a cell. Imagine that an enzyme creates a product, which also serves as the enzyme’s control molecule. This would allow a product to regulate its own production. Such circular schemes are known as feedback loops. A familiar feedback loop is the feedback between an audio speaker and a guitar pickup—when a rock guitarist holds her guitar against the speaker, the sound from the speaker causes the guitar strings to vibrate. This is picked up by the pickup, amplified, and fed back to the speaker, which now makes an even louder sound, vibrating the guitar strings even more. The result is an increasingly loud shriek, only limited by the power of the amplifier. This kind of feedback is known as positive feedback—a feedback where the product (sound, in our example) enhances the production of more product. Positive feedback also exists in cells—some enzymes speed up production in the presence of product. The result is a rapid, explosive increase in product (until the reactants run out). This can be useful if the cell needs to produce a chemical very quickly in response to an external stimulus.
More common is the opposite case: negative feedback. An example is Changeux’s L-threonine deaminase. This enzyme is part of a number of enzymes that work together to make isoleucine, starting from threonine. But isoleucine was also the control molecule that inhibited the activity of L-threonine deaminase. Thus, the product inhibits its own production. As a result, the enzymes make just enough product until the product molecules shut down further production. This is a nice way to control the maximum amount of a product molecule in a cell.
There are more complicated schemes that involve vast networks of interacting enzymes. The product of one enzyme may act as the control molecule for another enzyme, either enhancing or inhibiting its activity. The product of this second enzyme may again control the first enzyme, forming a two-enzyme feedback loop as shown in Figure 6.5, or the product may influence a third enzyme, which influences a fourth, and so on. Complicated schemes of feedback loops and mutual enhancement or inhibition provide the computing power that makes living cells seem intelligent.
FIGURE 6.5. A molecular feedback loop. The product 2 of enzyme A serves as a control molecule for enzyme B, while the product 4 of enzyme B serves as a control molecule for enzyme A. Depending on the control molecule’s influence on the enzymes (inhibition or enhancement), several programs can be implemented this way. In addition, substrates 1 and 3 may be the products of other enzymes, which themselves are controlled by control molecules. This way, more and more complex molecular programs can be constructed.
Rambling Across the Energy Landscape
Allosteric enzymes bring us closer to our understanding of molecular machines. A large change in the shape of an enzyme, when it binds to a substrate or control molecule, can be seen as a type of motion. We could imagine, for example, that this change in shape creates forward motion when we place an allosteric enzyme on a molecular track: Each time the enzyme changes shape, it pushes against the track and propels itself forward (Figure 6.6). Examples of molecular tracks include fibers and filaments, typically made of long strands of proteins. One example is actin, which we already encountered in Chapter 4. Another example is DNA— and indeed there are specialized machines that move along DNA like locomotives on a train track. We will meet some of these machines in the next chapter.
FIGURE 6.6. An allosteric enzyme bound to a molecular track. As the control molecule binds, the enzyme changes shape and pushes the enzyme forward along the track. However, the unbinding of the control molecule pushes the enzyme back to where it came from. To move forward, it would need to detach temporarily.
A molecular machine, however, is not simply an allosteric enzyme bound to a track. The problem with this idea is that an allosteric enzyme exists in exactly two states: It is in shape A if the control molecule is attached, and shape B when the control molecule is released. The enzyme may push itself forward when binding a control molecule, but would revert to its old shape and move backward once the control molecule is released. Just as with our ratchet in Chapter 5, the molecule would dither back and forth, but make no headway. To move forward, the machine needs to loosen its grip on the track during the back step and only attach during the forward step. But if the machine loses its grip on the track, the molecular storm will sweep it away.
Sisyphus at the Nanoscale
It seems we have returned to our original problem and must therefore add another step: The machine needs to reset itself to its original shape without creating backward motion. The machine needs a reset button, just like Maxwell’s demon. Remember, Maxwell’s demon could only work if he received external energy to erase his memory, that is, if he received energy to reset.
For a machine moving along a molecular track, the reset step would consist of detaching from a track temporarily, releasing the control molecule, and resetting the machine’s shape. Then the machine would rebind to the track, bind to a control molecule, and push forward against the track—repeating the cycle. This idea leaves us with a few questions: Why doesn’t the motion of the machine become randomized during the reset step? That is, why does the detached machine not get swept away by the molecular storm? And how does the presence of a reset step help explain how molecular machines avoid violating the second law?
To try to answer these questions, let us turn to Sisyphus, the mythical character condemned by the gods (for a certain divinity-angering faux pas) to push a large, heavy boulder up a steep hill for eternity. If he could get the boulder to the top of the hill, his penance would be fulfilled. Alas, his boulder was cursed: Every time he approached the hilltop, the boulder slipped from his hands and rolled back down. What makes pushing a boulder so difficult? When we push something up an incline, we have to use enough force to overcome friction and gravity. As we apply this force and move the boulder over a distance, we perform work (in physics, work is the product of force and distance), and doing work requires energy.
Imagine if Sisyphus and his boulder were denizens of the nanoworld. If the boulder were nanosize, it would be continuously pushed around by the molecular storm. Ignoring sideways motion, the boulder would be randomly pushed up and down the hill (more often down than up, because up requires more energy). Sisyphus could adopt a very energy-efficient strategy to get the boulder to move uphill: simply wait for the boulder to move uphill by itself, randomly pushed by the molecular storm. But this is no good—the boulder is more likely to be pushed downhill than uphill.
What if Sisyphus steps in the way of the descending boulder? He could block the boulder’s downward motion, while allowing it to move uphill (Figure 6.7). He could repeat this all the way up the hill, as long as he quickly steps behind the boulder each time it makes a jump uphill. Now we are getting somewhere: The boulder is propelled by the molecular storm, but this random motion is rectified by Sisyphus’s blocking its motion in the undesired direction. This is an extremely efficient way to move the boulder—a nano-Sisyphus would not have to push the boulder, and the boulder would move itself, driven by the molecular storm. Does this mean Sisyphus doesn’t need energy to move the boulder? No! Sisyphus still needs to move up the slope, step-by-step. These steps require energy.
How is this different from the ratchet in Chapter 5? The ratchet did not have a reset step and did not require energy. The ratchet was a passive device, which, by itself, was supposed
to rectify thermal motion. But Sisyphus is different: He rectifies thermal motion at the expense of using a source of energy to reset the system (stepping up behind the boulder).
Let us take this analogy further. When nano-Sisyphus steps up the hill, he converts some source of free energy into kinetic energy. Then he stops. His kinetic energy is turned into heat (his sandals are hitting the ground). The act of stepping up the hill degrades free energy into heat. Sisyphus and the ground are getting a bit warmer. Now, whenever the boulder tries to move downward, it bounces off Sisyphus (who is warmer), and some energy is transferred to the boulder (which is colder). In the end, Sisyphus and the boulder end up at the same temperature (thermal equilibrium), but as a result, some of Sisyphus’s free energy ends up moving the boulder uphill, even though Sisyphus is not actually pushing the boulder!
FIGURE 6.7. Left: A macroscopic Sisyphus is condemned to sweat as he haplessly pushes the boulder uphill. Middle: A nanosize Sisyphus wonders how he can utilize the random thermal motion of the nanoboulder. Right: Nano-Sisyphus comes up with an idea: Step behind the boulder as it descends, but let it move freely when it happens to go uphill.
Moreover, this free energy is ultimately degraded into heat, as demanded by the second law of thermodynamics. We have found a way to use the molecular storm to move the boulder without violating the second law. Without the chaotic motion of the molecular storm, the boulder would not move at all, and Sisyphus would be condemned to push forever. Yet, at the nanoscale, chaos can be turned into order, as long as we have a supply of free energy to periodically reset our machine.
Life's Ratchet: How Molecular Machines Extract Order from Chaos Page 18