# Why do so many biochemical reactions require enzymes?

Studying biochemistry as part of the Great Courses, I am struck that all 10 steps in glycolysis require an enzyme.

I’d have thought that evolution would have selected for a “simpler” pathway with fewer enzyme-catalyzed steps. If a reaction A + B requires an enzyme, three molecules have to be in the same compartment and collide instead of two.

My hunch is that having enzyme-catalyzed reactions instead of more reactive molecules reacting without catalyst gives cells the ability to control their metabolism, making homeostasis and regulation possible.

• Although it is more likely that 2 species will collide together as opposed to 3, the enzyme still helps. Enzymes are, if you go back to their definition, biological catalysts. They provide an alternate reaction route for the substrates, which has a lower activation energy. The increase in probability of reaction upon formation of an enzyme-substrate complex substantially outweighs the fact that 2 species are more likely to collide than 3. Without enzymes, digesting a meal would take weeks. Thankfully we can do it in hours. – arevmelikyan Apr 14 '20 at 16:04
• Control of the reactions may matter a lot. Many biological pathways are not just doing uncontrolled reactions but are regulating their rate and enzymes are complex machines that allow that, not just catalysts. – matt_black Apr 14 '20 at 16:23
• Arevmelikyan - well I know what enzymes do, it’s just seems more than a little coincidental that 10/10 steps in glycolysis require one. – Mike Apr 14 '20 at 17:01
• Martin - Random collisions and the evolution of enzymes for a specific job aren’t mutually exclusive. I don’t even really see how they are related. The molecules in the cell are still all moving about randomly, or at least chaotically. – Mike Apr 14 '20 at 17:10
• @Mike Don´t take it badly. I find the community here refeshingly honest and scientific. Most of the times. Sometimes you land on the bad side, and it´s sometimes not your fault, but the others have got up on the wrong foot in the morning. ;) – Karl Apr 15 '20 at 21:23

all 10 steps in glycolysis require an enzyme

We count it as 10 steps because there are 10 enzymes. There might be other steps that are fast (like a product acting as an acid or base) that we don't count because they don't need an enzyme.

I’d have thought that evolution would have selected for “simpler” pathways.

What is simple is that everything is happening in the same reaction vessel, the cytosol (well, there are more compartments, but just a handful). This is different from the organic chemist, who works with very reactive substances in pure form, carefully mixed in separate reaction vessels with distinct solvents refluxed at distinct temperatures for hours.

Is the very fact that enzymes are required make life more probable since the cells ability to control their quantities make homeostasis and regulation possible?

Yes, that is true. It also allows for a web of reactions rather than a single pathway (you can funnel fructose or galactose into glycolysis, and you can turn pyruvate into lactate instead of decarboxylating it to carbon dioxide and acetic acid).

Stable molecules require catalysts

One requirement for life as we understand it is a way to store, replicate and access genetic information. In our living world, that requirement is met by having nucleic acids. Under physiological conditions, RNA is fairly stable (good enough for a virus) and DNA is very stable (to the degree that sometimes DNA sample from extinct organisms preserve genetic information to this day). If these molecules do not form or decay on their own, there must be enzymes to act on them.

Granted, there could be other pockets of metabolism that work without catalysis. The other requirement, though, is to capture free energy to drive reactions away from equilibrium (e.g. to make the carriers of genetic information mentioned above). This is done through coupled reactions, where e.g. the free energy from food being oxidized (with oxygen as terminal electron acceptor) is captured in proton gradients across membranes and in the phosphorylation of ADP to obtain ATP. If any of the reactions going toward equilibrium would occur without catalysis, the free energy would be lost instead of captured by a coupled enzyme reaction.

General thoughts on complexity

In any complex system (government, computer chip, industrial process etc.), as the complexity grows, the number of steps that seem inefficient or over-complicated will also grow. Many of these steps might not make sense from the present view, but were put in place (or evolved) earlier to meet a historic need. Once in place in a system that has to run continuously, there is often no path to swap it out for a more efficient way of doing things.

One example from biology is the human eye, where the nerve cells run through the optical path, giving rise to the blind spot. The octopus eye "lucked out" and evolved in a way that makes more sense, but human eye sight is just fine (there is a "software patch" in the brain to fill in the blind spot):

Enzymes act as catalysts to increase the rate of reactions. In the absence of enzymes, most biological reactions would be incredibly slow, in some cases many many orders of magnitude slower than when catalyzed.

Given the large number of biological reactions that require enzymes, it seems that there might be an advantage to reactions that are slow in the absence of a catalyst, and that indeed seems to be the case. If the uncatalyzed reaction is very slow, an organism can control when the reaction happens by providing an enzyme at the desired time. When it is better for that reaction not to happen, the removal of the enzyme prevents the reaction from happening except at its slow uncatalyzed rate.

Since enzymes can be synthesized and destroyed in most organisms with a high degree of temporal and spatial control, the use of enzyme-catalyzed reactions provides cells with fairly good spatial and temporal control over metabolic reactions, thus optimizing the overall metabolism.

We speak of reactions that

• build complex molecules
• or break complex molecules at a specific position
• they must happen at constant 37°C
• all of which must be rather low energy
• or be very well controlled, like photosynthesis
• no fancy other reagents are available
• many would never happen at all without stringent control
• and/or the result could only be achieved in numerous reaction steps
• you don´t want side reactions
• cells are full of different compounds, all of which must be rather unreactive, or you´d get complete chaos
• you want the reaction to happen where you want it, and start and stop it
• you cannot control location of the educts, they diffuse in (and out of) your cell, because you have very little in the way of piping and valves

There is only one one way to get that: the self regulating production of self regulating catalysts. Enzymes. And a versatile energy source: ATP.

• Interesting point about the requirement that the compounds be relatively non-reactive lest all hell breaks loose. Thanks. – Mike Apr 14 '20 at 20:43

Life is all about a little compartment controlling the (bio-) chemistry inside it. From the moment the first cellular predecessors started forming in whatever the medium they had was, they had an ‘inside’ and an ‘outside’ and life evolved by taking control of the ‘inside’, protecting it against the ‘outside’ and then by some means, propagating the ‘inside’ to generate a ‘daughter inside’. They key concepts are self-assembly, autocatalysis and self-replication. To briefly introduce them:

• Self-assembly describes the spontaneous assembly of a more complex structure from simple starting materials by diffusion. For example, merely adding a zinc salt of a weak acid to a solution of protonated porphyrins will produce the corresponding zincporphyrin. If the porphyrin has an amino function at an appropriate position, these porphyrins will go a step further and dimerise spontaneously in solution. A more simple example that is frequently introduced in high school or undergrad courses are the hydrogen-bonded carboxylic acid dimers that form in aprotic solvents.

• Autocatalysis refers to a reaction where the reaction product is able to catalyse its own formation unspecifically. For example, consider the decomposition of cellulose acetate into cellulose and acetic acid. This reaction is catalysed by a proton source (acids), meaning that the more cellulose acetate degrades the more protons in form of acetic acid will be available and the reaction will progress faster. It is important to note that an autocatalytic reaction is typically unspecific: by adding sufficient amounts of a different acid (e.g. HCl), the reaction is also sped up and no rate increase is observable.

• Self-replication is probably the most important of the three: it describes the creation of an exact copy of a template from available starting materials. Usually, a self-replicating reaction will also be autocatalytic because with each synthesis a new template is generated allowing for the synthesis of additional individual units. Probably the most well-known example is the replication of DNA where a parent strand serves as the template for an exact copy which becomes the daughter strand. Note that enzymes are perfectly able to synthesise DNA without template—however, unspecifically.

While all three concepts are essential to developing life, the third one is the one that defines life and the possibility of evolution as we know it, because if you cannot generate an exact copy of yourself if you are not passing on yourself to a daughter generation. Furthermore, it becomes essential to restrict any possible replication to the desired self-replication. This is the beginning of the complex relationship between DNA, RNA and proteins/enzymes.

Probably the initial proto-cells depended heavily on the uptake of reactive predecessors to support self-replication from the outside broth but that isn’t a sustainable long-term solution. Thus, as time passed more and more reactions were taken under control to support the proto-cell in what it was doing, more and more simple enzymes developed until finally something resulted that could live on an energy supply and a carbon/nitrogen source alone—the first cells. These have a striking advantage over proto-cells that require the uptake supplies from outside so they probably rapidly prevailed and evolved further.

With every further step in evolution, the machinery became more sophisticated and enzymes became better and more specialised at their job. But more specialised also means more complex and more complex means easier to destroy. Thus, with the intracellular machinery becoming more and more complex it became more and more necessary to regulate every single little detail of what happens within a cell lest there be some way this could be harmful to the cells survival.

For example, a lot of reactions require acidic protons to be supplied to a certain functional group of the substrate. But for a complex cell it would be devastating to generate excess acidic protons and hope they find their targets because in another corner of the same cell an acid-sensitive substrate could be in the making or a basic environment is required to abstract a different proton. In addition, even if there were only one substrate that could react with this acidic proton, that substrate can still potentially react in more than one way with an undesirable pathway being more likely. All this is inhibited if the acidic proton is kept tightly in the centre of a catalytic pocket surrounded by the enzyme’s protein backbone, out of harm’s way.

All of this has lead to quite the opposite scenario from the one you paint: typically, a cell will have an enzyme available for every single reaction it wants to perform and leave nothing to chance or random collisions. Every single reaction that was left outside of an enzyme’s control has the potential to disastrous consequences and the cell is really better off limiting these as much as it can.

To exemplify this, I like to point to one of my favourite examples: Superoxide dismutase. This is an enzyme that catalyses the disproportionation of superoxide $$\ce{O2^.-}$$ generating diatomic oxygen and hydrogen peroxide. Superoxide is a highly reactive oxygen species that is generated in one-electron reduction of diatomic oxygen. This decomposition reaction is extremely rapid even in the absence of an enzyme: superoxide will disproportionate to the same products so rapidly that it is used as a chemical oxygen generator. However, even that rapid reaction is not fast enough for cells as even the short half-life of superoxide in water might cause irrecoverable damage. Thus, highly complex enzymes evolved and most species have a handful of them to combat superoxides. Furthermore, superoxide dismutases are remarkable in that they have one of the largest catalytic effeciencies (measured as the catalysed reaction’s rate constant divided by the uncatalysed reaction’s rate constant) $$k_\text{cat}/k_\text{background} \approx \pu{7e9 M^-1 s^-1}$$ – for a reaction whose background rate is already estimated at $$k_\text{background} \approx \pu{10^5 M^-1 s^-1}$$!

The product of superoxide dismutase, peroxide, is another highly harmful compound that has a high tendency to oxidise anything in proximity so it is also immediately broken down by peroxidases—but at least that doesn’t come as an equally big surprise since peroxide solutions are actually shelf-stable so peroxide has the far more obvious potential of harming half the cell.

Having considered all this dire need for controlling what actually happens in the cell’s inside, it should be less of a surprise that enzymes perform one step only and that each individual step of a biochemical pathway will have an associated enzyme—although it should be mentioned that if a reaction is close to equilibrium then enzymes will often indiscriminately catalyse both the forward and the backward reaction.

In the previous paragraphs I have not at all spoken of the idea of regulation: that a cell can turn on or turn off biochemical pathways as it sees fit. Bluntly, you don’t want to be breaking down your precious storage of glucose-6-phosphate (not actually stored as glucose-6-phosphate) into carbon dioxide and ATP if your cell is already oversaturated with ATP. The multitude of biochemical reactions catalysed by one specific enzyme each allows simple up and down regulation of individual pathways by means of inhibition or enhancing individual enzymes—another immediate advantage for the cell.

Now you might still want to argue why enzymes aren’t collected together to perform multiple steps at once or one after the other. Well—they are. Some of the most interesting molecular assembly lines are polyketide synthases. These multi-enzyme complexes typically consist of individual subunit domains which perform any of these steps: $$\ce{C-C}$$ bond formation in an aldol reaction typically using malonyl-CoA or methylmalonyl-CoA as a chain extender (ketosynthase), reduction of the resulting ketone (ketoreductase), elimination of water to create a double bond (dehydratase) and reduction of the double bond to give a saturated product (enoylreductase). Among these domains, there is an acyl-carrier protein (ACP) which serves as a robot arm transferring the growing chain from one domain sub-enzyme to the next. After having passed a domain, an acyl transferase will move the growing molecule on to the next one.

Multi-enzyme complexes are of course more difficult to synthesise and maintain as they are agglomerates of multiple individual enzymes. Thus, these complexes will typically only be assemblied where it is required, where an intermediate cannot be liberated or where a liberated intermediate cannot be sufficiently re-recognised.

• I'm glad the question was reopened to allow you to post this thoughtful and thorough answer! – Karsten Theis Apr 20 '20 at 21:55