The point of equilibrium in a process cannot be shifted by an enzyme under any circumstances. The explanation is simple: if an enzyme (or some other catalyst) lowers the activation energy for the reaction Y X, then it must also lower the activation energy for the reaction X Y by the same amount. As a consequence of this, an enzyme will hasten the rate of both the forward and backward reactions by the same amount, which will result in the equilibrium point of the reaction remaining unchanged. Therefore, even if an enzyme dramatically speeds up a process, it is unable to alter the pathway that a reaction will take.

Enzymes are responsible for directing all biological reactions in particular routes, notwithstanding the constraint that was just indicated. This is due to the fact that enzymes, which are normally only capable of catalyzing a single reaction, are exceedingly exact and selective in their actions. To put it another way, each enzyme precisely lowers the activation energy of only one of the countless chemical processes that its attached substrate molecules might go through. This is accomplished by the enzyme's ability to bind to its substrate molecule. This makes it possible for groups of enzymes to direct each of the numerous chemicals present in a cell along a certain pathway for reaction.

It is essential for the survival of living things that cells have the ability to synthesize a diverse range of enzymes, each of which have its own particular characteristics. Each enzyme has its own unique form, which has an active site. An active site can be thought of as a pocket or groove into which only particular substrates can fit. Because the enzyme molecules themselves are unaffected after taking part in a reaction, enzymes, like all other catalysts, can continue to perform their original roles even after the reaction has been completed.

An enzyme will typically catalyze a reaction that involves the participation of millions of substrate molecules per second. Therefore, it must be capable of binding a new substrate molecule in a timeframe of one millisecond or less. On the other hand, the amount of enzymes and the substrates that they use in a cell is extremely low. How is it that they are able to find each other so quickly? The molecular motions that are brought about by heat energy are moving so swiftly, which makes the possibility of rapid binding more likely. These atomic motions can be broken down into three distinct categories: translational motion, vibrations, and rotations. The process of a molecule moving from one point to another is referred to as translational motion. Atoms that are covalently connected to one another can be said to be vibrating when they move rapidly in and out of proximity to one another. Each of these events brings the surfaces of interacting molecules closer together, bringing about the desired effect.

To determine the rates at which molecules are moving, spectroscopy can be utilized in a variety of different ways. A large globular protein is continuously spinning around its axis at a rate of around one million times per second. The continuous translational motion of molecules also makes it possible for them to diffuse into the interior of the cell, which is a very efficient technique to investigate the space that is contained within the cell. Every molecule in a cell is subjected to this kind of collision with an incredible number of other molecules every single second. A single molecule in a liquid will move initially in one direction and then in another as it collides with and is repelled by other molecules in the liquid, producing a path that appears to be completely at random.

During a walk of this kind, the average net distance that each molecule travels from its starting point (as the "crow flies") is proportional to the square root of the amount of time that is involved. For example, if a molecule travels 1 meter on average in one second, it travels 2 m in four seconds, 10 m in 100 seconds, and so on.

Inside of a cell is a densely packed environment. In spite of this, research in which fluorescent dyes and other tagged chemicals were injected into cells has shown that small organic molecules are able to permeate through the watery gel that makes up the cytosol almost as quickly as they do through water. For instance, the typical amount of time required for a minute organic molecule to diffuse over a distance of ten meters is under one-fifth of a second. As a result, diffusion is an efficient mechanism for the movement of very small molecules over the very short distances found inside the cell (a normal animal cell has a diameter of 15 m).

Because enzymes move more slowly than substrates within cells, we can conceptualize them as being still because of this difference. The pace at which each enzyme molecule interacts with its respective substrate is going to be determined by the concentration of the substrate molecule. At a concentration of 0.5 mM, for example, there are a few abundant substrates that are present in large quantities. Due to the fact that pure water has a molecular weight of 55.5 M, there is approximately only one of these substrate molecules present in the cell for every 105 molecules of water. Even yet, the active site of an enzyme molecule, which is the part of the enzyme that binds to the substrate, will continue to experience 500,000 random collisions with the substrate molecule every second. When the concentration of the substrate is ten times lower, the number of collisions drops to 50,000 per second. This pattern continues until the collision rate reaches zero. In many cases, the formation of an enzyme-substrate complex takes place almost instantly after a chance encounter takes place between the active site of an enzyme and the surface of its substrate molecule. Now, a process that takes place really quickly can either form a covalent bond or break one that already exists. When one takes into account how quickly molecules can move and react, the rates of enzymatic catalysis that have been recorded do not appear to be all that remarkable.

Separation of two molecules can also be caused by the existence of noncovalent bonds between them. They form a number of weak noncovalent connections with one another, which continue to exist until the molecules are torn apart by random thermal motion and the connections between them are severed. In a general sense, the pace at which an enzyme and its substrate get dissociated is proportional to the strength of the binding between the two. In contrast, when two molecules collide and their surfaces are not well matched, they create very few noncovalent bonds, and the overall energy of association is negligible in comparison to the energy of thermal motion needed. Because of this, unwanted and unwelcome interactions between molecules that do not belong together are prevented, for as when an enzyme binds to the wrong substrate. This is possible because the two molecules in this situation separate just as rapidly as they combine.

Enzymes, despite the fact that they speed up reactions, cannot on their own induce energetically undesirable events to occur. To continue with our metaphor of water, enzymes cannot, on their own, cause water to move uphill. However, in order for cells to grow and proliferate, they need to do this specific function, which is to produce simple molecules that are then assembled into more complex and highly organized molecules. Enzymes are responsible for this, as we will see, since they directly relate energetically favorable processes, which release energy and produce heat, to energetically unfavorable reactions, which produce biological order.

How is it possible to quantify the term "energetically beneficial," and what does this term mean to those who study cell biology? According to the second law of thermodynamics, the universe always strives to be in the most chaotic possible state (largest entropy or greatest probability). As a consequence of this, a chemical reaction can only take place by itself if it results in an overall increase in the amount of disorder that exists across the cosmos. The topic of a system's free energy, which was covered in more detail earlier, is the one that best exemplifies this disorder that pervades the cosmos from a purely pragmatic standpoint.

The term "free energy," which is abbreviated as "G," refers to the energy that is available to perform work, such as igniting chemical reactions. This type of energy can be represented by the letter "G." The "reaction" that occurs when a compressed spring relaxes to an expanded state, releasing its stored elastic energy as heat to its surroundings, is an example of an energetically advantageous reaction on a macroscopic scale. The reaction that occurs when salt dissolves in water is an example of an energetically advantageous reaction on a microscopic scale.