Enzymes functions

Enzymes are proteins that play a maior role in the biochemical reactions happening every moment inside our bodies - everything from digesting a oowl of ramen noodles to flexing your muscles in front of a mirror Enzymes act as catalysts - meaning that they speed up the rate at which these biochemical reactions happen. So instead of waiting months to vears for a reaction to happen, it can happen in seconds - which is essential for life to happen.

magine trying to digest a single bowl of ramen for a year - you'd die of hunger before vou could do it Every biochemical reaction has a substrate and a product - so let's put them on this graph called a reaction coordinate diagram.

The X axis shows how a reaction progresses, while the Y axis shows the energy level at the different points along the reaction. In the beainning we've aot the substrate - let's call it A - with a fair amount of free energy

At the end of it. there's the product - or B, which ranks lower energy-wise. The energy of the product minus the energy of the substrate is called the energy of the reaction, also known as Gibbs free energy, or AG.

Because lower energy states are preferred, a reaction spontaneously occurs when the product has a lower free energy than the substrate - so a negative AG

So let's sav we're looking at one such spontaneously occurring reaction, but between going from the substrate to the product there's an intermediate transition step that has a really high energy state.

The amount of extra energy the substrate requires to get to the transition state - so the heiqht of the upslope - is called the activation energy - or a AGt plus plus. As soon as it enters the transition state, the molecule is highly unstable - and wants to go to a more stable lower-energy molecule

It either qoes back to being a substrate or to being a product

f it's a substrate once aqain, it can ao back up to the transition state if there's enough activation energy once more but if it becomes a product then it needs even more energy to get back to the transition state.

That's why over time, with millions of molecules doing this, the majority of substrate turns into product

Now, without an enzyme, the substrate might eventually harness enough activation energy to enter the transition state - but enzymes help speed things up quite a bit

Enzvmes are proteins that are folded in a particular way, so that they have a pocket called the active site on their surface.

When enzymes get involved in a reaction. the substrate binds to the active site, and together they form an enzyme-substrate complex, and that helps stabilize the transition state. So enzvmes decrease that extra energy requirement for the reaction - graphically turning our mountain into a hill.

Consider this analogy.

magine a little bov who's nervous about getting a vaccine - he's the substrate. and he turns into a vaccinated child - that's the product The transition state is where the needle goes in, and as vou can imagine - the oov might get really anxious and upset - a highly energetic and uncomfortable state.

In this scenario, enzymes are like adults who hold the bov and calm him down reducing the anxiety or energy level of the transition state and making the whole thing happen faster.

Fortunately, enzymes don't get used up in the process:

T hev attach to the substrate until it turns into the product and then release the product

As soon as thev're done, thev find another substrate

What's more is that enzymes and substrates are like biochemical soulmates - each enzyme is specifically designed for a particular type of substrate.

For example, amylase is an enzyme in vour saliva that specifically helps preak down large carbohydrates - into smaller sugar molecules that are then further broken down by other enzymes Now. the rate at which enzymes catalyse biochemical reactions is called enzyme kinetics, and there are two graphical wavs to look at this. The first. is the Michaelis Menten graph which has the concentration of the substrate, or [S1, on the X axis, and the speed, or velocity of the reaction or V which is how much product is formed over time. on the Y axis If there's a fixed amount of enzyme. the velocity of the reaction increases as more substrate is added - that is. until all the active sites on all of the enzyme become saturated

At this point, adding more substrate won't do a thing, because there's no more enzyme to bind it - so the speed of the reaction plateaus.

The point where the curve flattens out corresponds to the maximum velocity or Vmax, on the Y axis.

Now we can determine Km - which is the concentration of substrate at which the speed of the reaction is exactly half the maximum velocity

So we look at the Y axis. find what half of Vmax is, then we go parallel to the X axis until we reach our reaction curve. From there, we go straight down towards the X axis - and Km will be equal to that substrate concentration. The reason that Km is worth figuring out is that it inverselv reflects enzyme affinity - if Km is low, only a little substrate is needed for the reaction to skyrocket up to half of its maximum rate. so we're looking at an enzyme with high affinity.

On the other hand, if Km is high, then it takes a lot of substrate to get the reaction to go at half the maximum rate - so the enzyme has low affinity for its substrate.

Fundamentally, these graphs tell us about binding.

In fact, the same graphic representation could be used to show how strongly druqs bindina to receptors, or even transcription factors binding to DNA! So while we're here. let's look at how Vmax or Km can increase or decrease. Vmax depends on the number of enzvmes that can catalyse the reaction - so increasing or decreasing the number of enzymes will increase and decrease Vmax.

One way to increase Vmax is induction. which is when there's an increase in gene expression of an enzyme. The opposite - a decrease in Vmax - might happen if there's repression or silencing of gene expression. If we're talking about membrane receptors or transporters, up-regulation or down-regulation of receptors or transporters on the cell's surface would cause an increase or decrease in Vmax. respectively.

Finally, a drug might block

enzvmes, transporters or receptors non-competitivelv and cause a decrease in Vmax.

With non-competitive inhibition, the nhibitory molecule binds irreversibly to the enzvme active site, or reversibly to a different place called the allosteric site.

The net effect is that substrates can no longer bind to the active site and it is as if the number of available enzymes hao decreased.

So the effects of non-competitive nhibition are similar to what happens when there's less enzyme around, decreasing max. Now let's look at Km. which varies according to enzyme affinity. Affinity depends on the shape of the enzyme, and how well the substrate fits into the active site

So, let's say we alter the enzvme in a way that makes it bind more substrate - one way to do this is when a molecule called an activator binds to the enzyme and increases that enzvme's affinity for the substrate, lowering the Km.

Conversely, competitive inhibition can be used to decrease the affinity of the enzyme.

A competitive inhibitor binds to the active site instead of the substrate and usually doesn't get metabolized - plocking the enzyme

Now. since this is a reversible process, adding more substrate means that it's possible to eventually outcompete and displace the inhibitor.

But this increases the Km.

Now, practically speaking, a common way to change the shape of an enzyme is through phosphorylation by kinases or dephosphorylation by phosphatases and many processes are regulated this wav in our cells.

n fact. the terms desensitization and sensitization refer to changes in a receptor's shape which decrease or increase its affinity for a drug. Now, let's switch gears and look at the Lineweaver-Burke plot

This graph is based on the Michaelis Menten equation - which states that the initial speed of the reaction - or V0 - is equal to the concentration of substrate - S times the maximum velocity - Vmax, over the concentration of substrate + Km.

So here's where things get interesting ⁃ to get to our Lineweaver Burke plot we`re writing this equation down as a double reciprocal.

So flipping the whole thing upside down, we get that 1 over V0 is equal to Km over Vmax plus 1 over Vmax plus 1 over the concentration of substrate - [S].

Now we can use this equation on the olot: 1/V0 goes on the Y axis and 1/S goes on the X axis

Conveniently enough, after flipping the equation upside down, Km/Vmax is represented as a straight line, that intercepts both the Y and the X axes. Y intercept is equal to 1/Vmax, and X ntercept - since it happens negative to the oriqin of the axes - is -1/Km. Thus, the x- and v-intercepts can be used to quickly identifv Km and Vmax. The only trick is that, much like our reciprocal equation, now everything is upside down!

So processes like induction anc upregulation which increase the Vmax actually cause 1/Vmax to decrease so the line representing Km/max would slope lower on the graph than the control.

Conversely, processes like repression or downrequlation or even non competitive inhibition which decrease the Vmax, will increase 1/Vmax., makina the line slope hiqher on the graph than the control

So interpreting a Lineweaver Burke plot goes like this.

Stimulation makes the straight line appear to go down, inhibition makes it appear to shoot higher - it's, well, the reciprocal.!

Summary

Alright, as a quick recap.

Enzymes lower the activation energy required for the substrate to enter the transition state

Substrate binds to the enzyme at the active site, forming an enzyme-substrate complex, and it is released after it's transformed into the product.

There are two main wavs to look at enzyme kinetics: the Michaelis Menten graph, and the Lineweaver Burke plot Both can be used to determine the two kinetic variables, Vmax, or the maximum speed of the reaction, and <m, or the affinity of the enzyme for its substrate.