The Unexplored Role of Water in Biochemical Reactions
Proteins and Active Sites – A General Overview
Each of our trillions of cells are intricately designed machines which are uniquely suited to carrying out a highly specific set of tasks. The most important components of these machines are proteins, often referred to as the workhorses of biochemistry. The goal of my work is to explore the unique role that water molecules can take in the functions of these amazing biomolecules. Every protein is essentially a long chain of amino acids, anywhere from ten to ten thousand links in length, where each link interacts with every other to fold the protein into a specific shape, which gives it its unique function. Many proteins function as “enzymes”, which means that their job is to speed up (catalyze) certain biologically important reactions, such as the breakdown of other proteins. Reactions such as this could take thousands of years to happen under normal conditions, but in the presence of the proper enzymes, they can proceed to completion in less than a second.
The physical place on the enzyme where the catalysis occurs is known as the “active site”, and this is frequently located in a small pocket on the surface of the protein. This is because enzyme catalysis requires extremely specific conditions to be fulfilled, and controlling this is much easier in the confined environment of the enzyme interior than out on the surface. These close conditions create situations where molecules inside the active site have very different properties from those outside. Again, my research is focused on understanding how water is affected by the tightly regulated conditions of the active site.
If you think back to general chemistry, you might remember that water is a very polar molecule. What this means is that, even though water has an overall neutral charge (meaning it has the same number of protons and electrons), that charge is not distributed evenly over the molecule. The chemical formula for water, H2O, indicates that it has a central oxygen atom which shares electrons with two hydrogen atoms:
Oxygen is a highly electronegative element, meaning that it exerts a powerful pull on electrons. Because the two hydrogen atoms are less electronegative, the oxygen pulls much harder on their shared electrons, creating a situation where there is a disproportionate amount of negative charge centered on the oxygen, while the hydrogens have a disproportionate positive charge:
What does any of this have to do with enzyme active sites? The answer lies in the mechanisms by which enzymes catalyze reactions: they commonly use their amino acids to “push” and “pull” electrons, thus manipulating the bonds of other molecules. This requires the presence of many polar molecules, which, as you also might remember, interact powerfully with other polar molecules, such as water. As such, the polar environment tends to mean that water in the active site is most favorable when it is in a certain alignment with other amino acids.
This is important because it is almost entirely contrary to our understanding of how water functions at room temperature: the water in active sites is often locked into place, and hardly resembles liquid at all. My research is in understanding exactly how the confinement of this water affects its properties in the active site, specifically its ability to catalyze acid-base reactions (reactions which involve the transfer of a proton).
The Central Problem
Because enzymes are among the most abundant biomolecules, they present a seemingly simple way to answer the question of water confinement in the active site: simply investigate how the water behaves in a number of different actives sites (we’ll get into how I actually measure this later) with varying levels of water confinement, right? Unfortunately, this approach will not work because of several complicating factors. The first is that there’s no easy way to quantify how confined water is in a given active site, so this would be an imprecise measure at best. The second is that enzymes are unbelievably complicated and unique, so it is virtually impossible to find a pair of enzymes which have active sites with identical properties except for water confinement (remember: in scientific research it is crucial to isolate a single variable across your trials, so that you can make the strongest possible case for a causative relationship). How do we step across this?
Reverse Micelles as Model Systems
The lucky thing is that, while enzyme active sites are highly variable and hard to work with, water confinement is present in many different places. One easy to work with system is the reverse micelle. While this is a term that few will recognize, it is actually very similar to something that we all recognize: the soap bubble.
To understand it, we have to think back to our discussion of water. Remember that polar molecules interact strongly with other polar molecules. The flipside of this is that nonpolar molecules (in which the electrons are spread evenly across all the atoms in the molecule) are attracted to other nonpolar molecules. This is why simply running water over your dirty dishes doesn’t clean them – nonpolar molecules like grease and oil won’t dissolve in the polar water, and thus stay stuck to your plate. You overcome this by using dish soap. Here is a typical soap molecule, sodium stearate:
Notice that sodium stearate has two electronegative oxygen atoms on the right, while that long tail represents eighteen carbon atoms linked together, which is a very nonpolar structure. This gives sodium stearate what we call amphipathic qualities, where it has a polar end (the oxygen-rich head) and a nonpolar end (the carbon tail). This allows it to interact strongly with both nonpolar and polar molecules. Thus, the soap coats the grease and oil with its nonpolar tail, and the water is able to wash the soap bubbles away – with the grease inside – by interacting with the polar heads.
Reverse micelles are essentially these bubbles, but they have the proportions flipped: instead of a little bit of oil in a lot of water, they consist of a lot of oil with miniscule amounts of water. So what you end up with are oily solutions with tiny bubbles of our amphipathic detergent – called our surfactant – with the tails facing outward, and the heads facing inward towards a core of water:
Notice how the inward facing headgroups have negative charges and polar components to them, meaning that the water will interact with them similarly to the way in which it interacts with the polar amino acids in the enzyme active site. In the very center of the micelle we have water which behaves mostly as a liquid, but right at the edge of the water pool, called the interface, the water displays the same non-liquid structure as water in active sites does. Now we have a way of replicating the confined water!
Now we have to ask the question of how we adjust the level of confinement within the reverse micelle water core. The answer is surprisingly simple: by varying the proportions of surfactant and water present in solution, we can alter the size of the micelles, thus varying the proportion of the water which is present at the interface region. Smaller micelles mean that a larger portion of the water is found at the interface, meaning that the pool has a greater confined character.
Photoacids Inside the Micelles
Now notice that while we have found a way to vary the water confinement in our model system, we aren’t just interested in the confinement itself, but in how it affects the dynamics of reactions within the system. The reaction that we’ll be looking at is a simple acid-base reaction, which you might remember looking something like this:
In this reaction, the acid (A) donates its proton to the water, giving it an extra hydrogen and a positive charge. This is an extremely commonplace reaction in biological systems, and it directly involves water, making it a perfect subject for my research.
Now we face one last complication: we are interested in how fast these reactions happen in the presence of confined water, so we need a way to actually see the reaction and how fast it moves. This is incredibly difficult, as moving a single proton is a virtually instantaneous process, which can take only nanoseconds. For reference, a nanosecond is the length of time in which something moving at light speed would travel about one foot. How do we observe something so fast?
The answer lies in a particle called a photoacid and a technique called time-correlated single photon counting (TCSPC – don’t worry, I’ll explain it). A photoacid is, simply, a molecule which is normally not acidic, but when struck with the right wavelength of light, becomes an acid and donates its proton to a base, in this case, water. With a photoacid, we can use light as a kind of “starter pistol” for the acid base reaction; the acid won’t begin the proton transfer process until we hit it with a high energy light pulse.
TCSPC is somewhat complicated, but can be broken down relatively simply: it’s a really fast laser that fires millions of pulses a second. Striking the photoacid-in-micelle solution with the laser excites it, initiating the proton transfer event. Typically, when a molecule is excited by photon of light it will then release another one shortly afterwards. The properties of the released photon are slightly different depending on whether the photoacid has already donated a proton or not. TCSPC just involves striking the sample with the laser and recording all the little flashes of light that are given off, allowing us to quantify how quickly the AH form becomes the A– form. We can then compare the speed of this reaction correlates with the micelle size (the degree of confinement of the water), to get a sense of how the reaction might proceed in a confined biological environment.
Confined Water and Acid-Base Kinetics – Why Should We Care?
If you’re still reading at this point, you probably have a major question on your mind: what could possibly be the reason for wanting to know any of this? The reason lies in the incredible intricacy of biological systems. One growing field, for example, is de novo protein design, in which scientists attempt to create their own proteins to carry out specific functions. Because of the incredible complexity of these systems, one needs to understand every aspect of their system, including the dynamics of water molecules within the active site.
Protein design has enormous potential in many fields, from medicinal purposes to industrial purposes. For example, a designed protein might be able to target a specific strain of bacteria with perfect efficiency by interfacing with another protein unique to the infectious strain, thus leaving our own cells and our microbiomes untouched. Designed proteins are promising for industry and chemistry as well, as many enzymes carry out their assigned tasks with efficiency and specificity that human chemists can only dream of. Harnessing that power might even open the doors to converting atmospheric carbon dioxide into oxygen – something plants do every day – or breaking down plastics and styrofoams which ordinarily break down over thousands of years.
Author Bio: Alex Poblete is a senior majoring in biochemistry. He is currently working on his Honors thesis with Professor Kana Takematsu, studying the physics of how water behaves in biological systems such as enzymes. He enjoys reading, playing the piano, and working as a tutor in the Center for Learning and Teaching.