Big Picture:
You should review this figure on

Remember that oxidation/reduction (RedOx) reactions involve electrons being traded from positions of high potential energy to ones of lower potential energy, with the resulting release of some free energy (the difference, or delta) between the start and final state.
The overall reaction in respiration combines carbon and hydrogen gotten from carbohydrates and other carbon-containing molecules with oxygen to make CO
2 and water. But, instead of doing that all at once, there is a long series of reactions releasing smaller amounts of free energy, some of which is captured in nice, “bite-sized,” chunks of energy we can use. What “bite-sized” chunk really means is electrons in compounds easily oxidized. The two main bites of energy for the cell are ATP and NADH.
We will produce ATP through simple reactions called “substrate level phosphorylation” and through oxidative phosphorylation. In this second process, NADH is the source of electrons for the electron transport system, which uses RedOx reactions to pump protons to the inter-membrane space. Finally, the ATP synthase uses that proton gradient to synthesize ATP.
We will start at the end, because it makes more sense this way.

ATP Synthase

Enter the ATP synthase:
Atp_synthase This machine is almost too good to be true. It’s in the mitochondrial inner membrane, the chloroplast inner membrane and the plasma membrane or inner membrane of bacteria. The protons flow down gradient through a channel, which leads to mechanical changes that spin the rotor (more like a ratchet). This mechanical energy is then used to drive ATP synthesis. Thus, we convert mechanical energy to chemical potential energy. The process is also called “chemiosmosis.”
Too cool. Here is a video. It’s not the one from the creationist site.

All this needs to run is a proton gradient. How do you get that? Well, that depends. In the mitochondria and chloroplast, the proton gradient comes from a system called “Electron Transport,” also taking place in the inner membrane of the mitochondria (or chloroplast).
Electron Transport is a series of RedOx reactions taking place in or near the membrane that result in protons being removed from various chemicals and released into a confined space between two membranes (such as between the inner and outer membrane of the mitochondria).

Reducing equivalents or “Reduced Cofactors”

To run electron transport, you need electrons in a high energy state. That is, you need something that has electrons that are relatively easy to remove to a lower state. The thing donating the electrons is the “reducing agent” (it gets oxidized) and therefore we call it a “reducing equivalent” or “reduced cofactor.” It will be one or more “B” vitamin derivatives in its reduced form.

A word about “cofactors.” I hope all of you were starting to see the patterns in the nucleotide cofactors. Take a look at the separate entry on cofactors if you like.
NAD clearly has a lot in common with ADP. NADH has particularly high RedOx potential and is sometimes used directly in enzymatic reactions requiring free energy. These really are variations on a theme. The cofactors in electron transport are variations on another theme, or, pardon the pun, variations on a
Heme. These are the iron-containing rings that you are familiar with from Hemoglobin, which can carry oxygen in your blood. But, similar cofactors will be used in electron transport.

If you want to carry electrons in a membrane, you need something that is hydrophobic to do it. The go-to cofactor there is “Coenzyme Q.” It is not based ADP. The “MeO” in the structure stands for a Methyl-Oxygen or “methyl ether.”
You will see these structures over and over. They will always be doing similar tasks.

Oxidative phosphorylation.

Technically, this includes two stages: the phosphorylation by the ATP synthase we’ve already discussed, and the
Electron Transport System which uses oxidation/reduction reactions to move protons to the inter-membrane space to drive the ATP synthase.
I stole the figure below from a great text book called “Molecular Cell Biology.”

The key players here are called either electron carriers such as Cytochromes and cofactors such as coenzyme Q, or oxido/reductases, enzymes that catalyze the transfer of electrons, frequently while pumping protons across the membrane. You don’t need to know the names of them, though I might use them. The complexes are just labeled with Roman Numerals.
The cytochromes are small-ish proteins that contain bound within them, usually, an Iron atom/ion. This is cytochrome c, one of the most well characterized cytochromes.
The multi-ring structure in the middle is not part of the protein per se. It is called a “heme,’ as in “hemoglobin,” and is held in place by the protein. The red dot is an iron.
Technically, the heme is not a cofactor, but a “prosthetic group.” That is fairly subtle distinction. A cofactor generally enters the active site of a protein when it is needed, is altered, then has to be replaced. A prosthetic group stays in the protein continuously. Below are two images of a heme chelating an iron.
Look at the general scheme on page 171, figure 9.13. Electrons are passed from NADH or FADH2. These enter the “electron transport system” which is the string of cytochromes and other acceptor/donor molecules in the inner membrane. The electrons pass down through a series of RedOx reactions, the acceptor for one step being the donor for the next. Finally, the electrons are passed to Oxygen, which pairs with protons to make water.
At three steps along the way, just due to the orientation in the membrane, the H
+ product of the dehydrongenation (oxidation) is spit into the intermembrane space (between the two membranes—we’re so clever with names). This leads to an electrochemical gradient.

Key Points:

  1. The donor of electrons are cofactors NADH and FADH2 .
  2. The oxidation/reduction reactions pass electrons from one carrier to another in or near the inner membrane.
  3. The reactions result in protons being pumped to the inter-membrane space, establishing the electrochemical gradient that runs the ATP Synthase.
  4. The final recipient of the electrons is oxygen, which combines with H+ to make water. That’s why you breath out water.
The remaining question is: Where did the reduced cofactors NADH and FADH2 come from? We’ll get to that next.

Citric Acid cycle

this is also known as the tricarboxilic acid cycle (TCA cycle) or the Krebs cycle, after the person who worked it out.
Yes, I know all the steps and where they connect to the rest of the BSC (Big Scary Chart). No, you don't have to. It occupies the very center of the BSC, for good reason: Even though it is always taught as though it is solely part of respiration, it is really central to all the metabolic pathways. The things made in this cycle are used as building blocks for many other things.
The first thing you need to know about it is that it is a
CYCLE as the name says. What that means is that the pathway accepts new carbon units at the top, goes through a mess of steps, and then regenerates the starting molecule, which is ready to accept carbon units again.

The other key things are:
It takes place in the "matrix" or center compartment of the mitochondrion
It oxidizes carbon at several places, which releases CO
2 gas…this is the reason you breath out CO2
The electrons taken from the Carbons in this step are passed to the nucleotide cofactors we saw in the Electron Transport System (ETS) today. NADH and FADH
2 cary these electrons to the ETS

So—you need a proton gradient to drive the ATP synthase (chemiosmosis). That proton gradient could come from anywhere…but in all the cases we will examine, it comes from ETS…a series of RedOx reactions taking place in or near the inner membrane of the mitochondrion and passing protons to the inter membrane space. To run this, you need high energy electrons. Where do you get them? Well, could be anywhere…but in this case, it's electrons taken from carbon atoms in the Citric Acid cycle and carried to the ETS by NADH and FADH
Where does the new carbon come from? Well, in this case, from the food you eat. Could be glucose. But could also be any other carbon- and oxygen-containing food molecules, which is all the things you eat that are actually food.
That's really all the important stuff. Now for some more details.

This is pyruvate,Pasted Graphic . It is an end product of breaking down glucose (glycolysis). But, it can also come from other places. It has a carboxyl at the end (that thing at the right). Actually, it is de-protonated in this form, which is why it is pyruvate instead of "pyruvic acid. The carboxyl gets removed as CO2, a “waste product.” That’s an oxidation and the thing that gets reduced is NAD+, yielding NADH + H+ (NADH plus a free proton). As part of the same reaction, the remaining acetyl group (the remaining two carbons with a C=O on the end) is transferred to the cofactor “Coenzyme A” or “CoA” for short. We looked at that a little. Acetyl CoA will be an important source of building blocks for other things. Here, it is used to transfer the acetyl to a 4-carbon molecule called oxaloacetate (Remember "OAA" in the BASS 2 activity?) Here it again is shown in its protonated form.Oxaloacetic-acid-3D-balls. Adding two carbons to the 4-carbon OAA makes Citrate (or "citric Acid), a tricarboxylic acid: Citrate-3D-ballswith a total of 6 carbons. Here it is shown deprotonated with resonant bonds from the carbons to the oxygens)
This is going to go through a series of steps that will regenerate oxaloacetate. A CO
2 molecule will come off in both step three and four. Again, this is a net oxidation of the carbon. The oxidizing agent (the thing reduced) is NAD+, yielding NADH plus a free proton. Step five is called "substrate level phosphorylation" producing GTP. All this means is that there is a simple kinase reaction…but it is running in the backwards direction to what we usually think of. Normally, kinases take a phosphate from ATP and transfer it onto some target. In this case, the enzyme takes a phosphate from solution and transfers it to GTP. In the book, it looks like this immediately transfers its phosphate to ADP to make ATP. It can. But GTP is useful itself in many places. Keep that in mind. GTP will be a major player in regulating enzymatic pathways. Two more steps of import: reduction of FAD Flavin_adenine_dinucleotide(Flavin adenine dinucleotide...the flavin (top left) is not technically a the name is not quite right. ADP is the bottom right half). It works much like NAD. It gets reduced (gains electrons) and can spit them back out into Oxidative Phosphorylation. Finally, there is 1 last reduction of NAD+ to NADH + H+, regenerating oxaloacetate.

Glycolysis…we'll cover that next.

You may notice that many of these steps require magnesium ions. We should talk some time in general terms about the role of cations in enzymology.


Key steps in glycolysis are:
Hexose Kinase (1st step), product is glucose 6-phosphate, keeps glucose inside cell and keeps gradient “downhill” into the cell. This uses an ATP, but is not regulated.
2nd step rearranges it to fructose 6-phosphate. That is, the hydroxyl of position 2 oxidizes the carbonyl on position 1. Position 1 gets the H to make a hydroxyl there and position 2 becomes a carbonyl. The enzyme can be called “hexose-phosphate isomerase” or, as it is on the diagram, phosphoglucose isomerase.
This next step is the rate-limiting and tightly regulated step. Phosphofructokinase (3rd step). This adds a second phosphate to fructose 6-phosphate, making fructose 1,6-bisphosphate. The regulation is via an allosteric site that binds ATP. Now, you have to know two things here: the enzyme has 2 sites that bind ATP, one in the active where it is necessary as a substrate for the reaction and a second that inhibits the reaction; the first site binds with higher affinity than the second. So, if the concentration of ATP is low to average in the cytoplasm, only the active site binds ATP. However, if the concentration of ATP gets high, indicating that more ATP is really not needed, the second, inhibitory site becomes occupied and shuts the enzyme off.
Aldolase (4, clips the six-carbon sugar diphosphate into 2 Triose phosphates called dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (G3P). G3P is the more useful one and the two are freely interconverted by triose isomerase (there are those who call it…"TIM?"). Since there are two, G3P made, all the subsequent steps can be done twice for each glucose).
The next step is carried out by Triose Phosphate Dehydrogenase (takes hydrogen off a triose phosphate). Some charts label this "Glyceraldehyde phosphate dehydrogenase." Glyceraldehyde phosphate is a triose phosphate.
If something is “dehydrogenated” it is the same as being oxidized. The thing that is reduced is NAD
+. If you are interested in the chemistry here, it is kind of cool. Phosphate free in solution enzymatically attacks position 1, the carbonyl. The exchange results a carboxyl with a phosphate on it. That’s a really high-energy phosphate. Hydrogen from the carbonyl carbon is transferred to NAD+, reducing it. This is the first energy payoff in that it creates two NADH + H+ (one for each of the G3P), which can be used in oxidative phosphorylation or as a high-energy cofactor in certain other reactions. But, as we'll see later, this reduction can present a problem.
The phosphate on position 1 doesn’t stay long. It is transferred to ADP to make ATP in the next step. This process is called “substrate-level phosphorylation,” and is carried out in this case by the enzyme phosphoglycerate kinase. Those of you learning how the names work might say: “Wait…shouldn’t that name mean it ADDS a phosphate to 3-phosphoglycerate?” How clever of you to notice. The enzyme is named for the reverse reaction to the one we are talking about as part of glycolysis. There are several key enzymes for which this is the case. It turns out that if you purify it away from everything else, it seems to favor the reverse reaction. It can drive it either way. The reason the reaction goes primarily toward producing phosphoglycerate is because there IS a next step. You see, as soon as 3-phosphoglycerate is formed, it gets pulled into that step.
Next is a simple rearrangement where the phosphate gets moved from position 3 to position 2.
2-phosphoglycerate then undergoes a step carried out by a fairly famous enzyme: Enolase. This enzyme pulls a water molecule out but makes the most unstable, highest energy molecule in the series, Phosphoenolpyruvate (PEP). Enolase is essential in every cell that does glycolysis. It is rather famously inhibited by fluoride ions. This is why we put fluoride in toothpaste (and, in less conspiracy-minded communities, in the water). The fluoride kills the bacteria in your mouth. Anyway, that phosphate next to the double bond has to go. It's way too unstable. The enzyme that does that is another really well known one called pyruvate kinase (again, named for the favored reverse reaction). It takes that phosphate and transfers it onto ADP to make one more ATP (well, two more, because we start this portion with two triose phosphates). The product is pyruvate, which goes off to the mitochondria for the TCA cycle.

Enzyme Cofactors


Wikipedia defines this as a “non-protein chemical compound that is bound by a protein and is required for the protein’s biological activity.” That’s a pretty good explanation.
When I was your age (have you ever once heard a story you ended up liking that started out like that?), these were called “coenzymes” and a few of them still have that name (Coenzyme A and Coenzyme Q come to mind). But, “cosubstrate” is really a better term for what they do.
They are called “small molecules” because, compared to proteins, they are small. However, some are huge compared to simple molecules you remember from chemistry.

Some Names:

If an enzyme uses a cofactor (most do), the protein part without all the other necessary components is called the “apoezyme.” The form that includes the protein part(s) necessary plus all other cofactors is called the “Holoenzyme.”

ATP and other Nucleotide cofactors

The most common cofactor is ATP, which you think of as a source of free energy. As you know, it is usually used to transfer phosphates onto something else. It is in the category known as “nucleotide cofactors” because, as you should recall from the section on DNA and RNA, the building blocks of RNA and DNA are called nucleotides. ATP is also a building block of RNA.

One very common group of cofactors that will be important in the upcoming weeks are those involved in Oxidation/Reduction reactions as either donators or acceptors of electrons. For example, when the enzyme Alcohol Dehydrogenase converts alcohols to aldehydes, it is an oxidation (removal of electrons). Something must take those electrons. The enzyme passes them to a cofactor called NAD+ (it stands for “nicotinamide adenine dinucleotide”) and it is basically niacin (vitamin B3) attached to ADP. ADP is the bottom thing, Niacin is the top thing.


A cofactor with a very similar role in metabolism is FAD (Flavin adenine dinucleotide). The flavin is on the top. It’s not really a nucleotide...but, whatever. Once again, it’s clearly attached to ADP.

Cofactors are also used to help transfer groups of carbon, carbonyls or carboxyl, amines or other functional groups these are essential for building the complex molecules we need.
The most common one is Coenzyme A (or Co-A). It looks like this:

Do you recognize ADP again (actually, it’s called ADP-Phosphate because of the phosphate in the 3' position of the ribose). Attached to it is a small peptide-derived portion with an SH on the end (sulfhydryl). Recall those are good reversible oxidation/reduction sites. That site accepts the acetyl (two-carbon organic acid) groups. This is important in something called the Citric Acid Cycle we will learn about soon.

Do you see some patterns? Good.

Signaling intro

This is the simplest slide of cell signaling that has enough information to be useful. Start by looking at the types of signals that get to the cell (growth factors...survival factors...death signals” look at the type of receptors available and the way the messages leave the membrane area. Look up “Receptor-tyrosine kinase” either in the book or online just to pick an example of how a signal cascade starts off.

Signal Overview
  1. Here is a link to the signal talk I gave a while back, which includes some discussion on G proteins.


Cell communication takes many forms, but there are commonalities in most of them. Cells may communicate by direct contacts between proteins on their surface, but do not need to be in physical contact for most events. There are lots of examples of "distinctions without a difference." What I mean by that is that you will hear reference to endocrine or hormone signaling (signal molecule made in a gland and secreted into blood), autocrine signaling (signal molecule made by same cell receiving it), paracrine signaling (signal molecule made by cells nearby the receiver), neurotransmitters, cytokines, chemokines…there really is no difference in how any of these work. It's like asking "Did you drive in your car to get milk or walk to the store?" Sure…there are differences…but the milk is the same.
In general, there are three steps:
  1. A signal molecule, or “ligand,” usually soluble (but can be bound on another cell or on the extracellular matrix), floats up to the target cell. The signal molecule may be a small protein (such as insulin), or a much smaller molecule (such as epinephrine). It binds to the extracellular face of a receptor, a protein that spans the membrane of the target cell
  2. The receptor changes shape (conformation) when the ligand binds. This is no different in concept to the idea of allosteric changes to proteins we discussed when we talked about inhibitors of enzymes. The tertiary structure of the protein changes. These changes affect not only the outside face of the receptor, but the internal side as well. This is the transduction event. That’s how the information gets through the membrane.
  3. The change in shape on the inside of the receptor leads to some change in the biochemistry of the cell. It may make available a new binding site, or a new active site. But some change inside the cell happens and ultimately, there are changes to how the cell behaves. There is a nearly limitless set of possibilities. Frequently, some enzyme gets activated and makes something called a “second messenger.” In this way the signal is amplified and may have many effects in the cell.
  4. (so much for "three steps") Eventually, the cell has to stop responding. The pathway has to have a way to shut down. The ligand may release from the receptor, but that only stops new signal events. Enzymes that were activated need to be deactivated. Second messengers made need to be destroyed.

More specific

We have looked at a small subset of G-protein-coupled receptors. These are among the oldest and most diverse types of signal molecules. We won’t look at all the possibilities, just one or two.
There is a decent animation of G-protein signaling
here. The structural representation is not so good. But, it’s not too bad.
Here is a link to David's "molecule of the month"
blog on the beta adrenergic receptor.

The key points are that:
  • G proteins are inactive when GDP is bound, active when GTP is bound.
  • Turning them “on” requires exchanging the GDP (kicking it out) for a GTP (allowing it to enter). This exchange is facilitated by the receptor, once ligand is bound.
  • G proteins are molecular timers. Once turned on, they will turn themselves off by hydrolyzing the GTP to GDP.
  • While they are in the “on state,” they activate other target proteins, usually enzymes, which then generate second messengers.

In addition, if you have time, there is a wonderful lecture by Bob Lefkowitz, the discoverer of the beta andrenergic receptor (and the first GPCR other than rhodopsin) that can be found
here. It’s long (50 minutes, but may be worth class time to see and discuss). He does a great job of discussing out the experiments to show how it works were done.

You may notice that there are many other animations for G-protein coupled signaling at You Tube. Some of them open ion channels (as in nerve cells), some of them connect to adenyl cyclase, as we have discussed. Others connect to other pathways, such as the phospholipase pathway.
The cAMP is the “second” messenger that goes off and activates many other proteins in the cell. Because each cell will have its own cAMP-responsive proteins, each cell may respond differently to the same signal molecule. As we discussed, there also will be, potentially, more than one receptor for a given ligand, resulting in different pathways being induced.

How is the signal turned off? Well, ligand may leave the receptor..but that doesn’t stop everything that is happening away from the receptor. The G-protein is still active, as is the adenyl cyclase and there is a lot of the second messenger, cAMP still floating around. The G-protein turns itself off by hydrolyzing the GTP back to GDP and lets go of its target protein (in this case, the adenyl cyclase) That turns off the target protein. Well…you still have all that cAMP around.
There is another enzyme that destroys that, converting it to AMP (not cyclic anymore). That enzyme is phosphodiesterase, often abbreviated PDE.

Below is a list of common first and
"second messengers." You may run into some of them.
Screen Shot 2016-09-29 at 10.05.45 AM

Enolase: an example mechanism

I Thought I would take a minute to talk about a reaction mechanism. This is not something on which I would test you. However, I thought getting into the mechanism…even just the idea of a mechanism…would be useful.

What is a “mechanism” anyway?

Briefly, a mechanism is a detailed (as detailed as possible) description of the steps of a reaction. You got a little into it last year with the idea of an elemental step in kinetics. However, with the advent of crystal structures of enzymes, more details can emerge.
To review, we have talked about protein structure and the fact that the overall structure results in positioning particular reactive groups of the side chains in a position to carry out chemistry. I’ll add here that metal ions are important factors in the function of many enzymes. Many proteins coordinate metal ions in their active sites and the metal plays an important role in the chemistry (for now, think of “coordinate” as “bind”).
First, some images to get oriented. This is the protein molecule shown in familiar “ribbon” representation.

I’ve shown a dimer of two identical proteins. You should see a blue dot, which is the magnesium ion. On the right, you can see a couple of the amino acid residue side chains that interact with the ion. We’ll zoom in on that region here:

You can see that the positively charged metal ion is being held in place by several side-chain oxygens. I’ve labeled some of them (two aspartic acids, “D,” and one glutamic acid, “E”). There are others.

Now, I’m going to add in the substrate, 2-phosphoglycerate. I’ll make it solid, and I’ll highlight a couple of side chains that interact with it, Lysine 345 (K345) and glutamic acid 211 (E211). At the other end of 2-phosphoglycerate, you may see how close the metal ion is to the oxygens of the carboxyl end. The electron-poor magnesium ion is right up against that electron-rich oxygen. Now, normally, oxygen wins the battle for electrons easily...and it does here too. But the whole structure draws electrons away from a bond with a hydrogen at position 2.

Below is a schematic of the reaction of enolase to form PEP.

The arrows in the diagram represent moving electron pairs. Here’s what happens:
The metal ion, which is 2+, is withdrawing electrons from the two oxygens, there on the left (this version of enolase has two magnesium ions). Lysine has a basic nitrogen. You see the lone pair depicted on the nitrogen. This makes the H attached to Carbon 2 behave like an acidic proton…not normally the case. However, the proton leaves carbon 2 and gets the electrons in the lone pair on lysine’s nitrogen, the electrons that were in the C-H bond move over and form a double bond with C1. That makes it an “alkene.” The electrons from the double bond of the C=O will flip out to that oxygen, making it negative. The carboxyl of Glu211 is making a hydrogen bond with the OH on C3.
In the second panel, the OH from position 3 will combine with the acidic proton from Glu 211 to form water. The elections from the other carboxyl oxygen will form a double bond, the electrons from the 1-2 double bond will move to the 2-3 position. The product, PEP, is released (Phosphoenolpyruvate). The extra H from the amine of lysine 345 (picked up from carbon 2 in the second panel) transfers over to Glu211 regenerating the enzyme in its starting configuration.

See…kind of cool.

Intro Enzyme Kinetics

Enzyme Kintetics

Most of the images for this article were taken from the Wikipedia entry on Enzyme kinetics. You can read that here. It gets a little intense, even for our purposes.
Some Words:
Enzyme: a biocatalyst that acts to speed up the rate of a reaction, often by many orders of magnitude (that is, many factors of ten). Most enzymes are protein, though some important ones are RNA.
the starting material acted upon by the enzyme to make the product.
Active site: the site in the enzyme in which the substrate binds. It is the site at which the catalysis takes place.
Saturation Kinetics
We’ll call the above graph, “fig 1.”
Notice firstly that the graph is determined from a series of kinetic experiments and that Y axis is a rate (usually in mol/L*s that is change in concentration over change in time). So, each point is from an experiment in which you had an initial concentration of substrate and then measured the change in that concentration versus change in time (the initial rate). The points on this Fig.1 are each the initial slope of one experiment from Fig. 2, below.
The image below was taken from Campbell’s website.
EnzymeRatesFig 2
It represents three experiments tracking a reaction where A B and following the rate of disappearance of A. The three experiments have different initial concentrations and different initial rates. The slope of each of those lines becomes a point on Fig 1. The sign of the slope is changed so that it is positive (remember from chemistry that Δ[B]/Δt = -Δ[A]/Δt).
Experiment 1 above might be one of the high points on Fig 1. Experiment 3 would be at lower concentration.
Saturation Kinetics:
The pattern seen in Fig. 1 is called saturation kinetics for reasons that should be obvious.
Please Note: the flat part of Fig 1 is not when the rate of the reaction is zero.
It shows that above a certain concentration of "A," the initial rate no longer increases. The reason for this is that all the enzyme molecules are occupied. Adding more substrate just adds to the “line” of substrate waiting, so to speak (see the “Starbucks Model below). Saturation kinetics are also called “Michaelis-Menten” kinetics after the people who first explained it. You should know that almost no enzymes follow this model exactly. It also deals with a single substrate...but can be adapted to deal with more. Nevertheless, it is an important model that helps explain a lot. The idea is that there are at least two steps to the overall reaction: Binding of substrate to enzyme; and catalysis.
You can think of the process of catalysis as proceeding in at least two steps (there are usually more): a binding step and a catalytic step.

“E” is enzyme, “S” is substrate, “ES” is enzyme bound to substrate and “P” is product.
There is an “on” rate and an “off” rate (k
1 and k-1) indicating that a substrate molecule can bind and then come off again. The second step is determined by something internal to the enzyme: its catalytic constant. Note that if I blocked the second step, the binding step would come to equilibrium, with substrate binding and releasing at equal rates. Under those conditions, I could calculate a “KD,” for “dissociation constant,” which would be k1/k-1. This would represent the concentration at which you would see ½ maximal binding.

Important Points on Figure 1.

There are two important points on Fig. 1. We have already talked about the first, V
max. That is, the maximum velocity, or rate, of the reaction once all of the enzyme molecules are busy.
The other point is a substrate concentration known as “K
M,” which is the concentration at which the rate is ½ of the Vmax. Note that if the catalytic step is fast and we are still in the linear portion of the graph, the KM is approximately equal to the KD.

High or Low Substrate concentrations.

Imagine two situations, one in which substrate concentration is low and one in which it is high.

EnzymeCartoonFig 4.

In the first case, the slow step (rate-determining step) is the binding (first) step. The catalysis step may seem instantaneous, by comparison. This gives us the steep initial portion of Fig. 1. The rate is determined by the rate of binding.
In the latter case, essentially all the enzyme molecules are occupied. No enzymes are sitting around idle. Thus, the reaction is going at its maximum velocity (V
max), the (nearly) horizontal portion of the graph at the top. At that point, the overall rate is governed by the catalytic step, give by k2.

The Starbuck’s Model

Think of the reaction being catalyzed as production of cappuccini at Starbucks. The enzyme is the “Barista” and the customers are the substrate. If you count cappuccini per hour, you get a rate. If there is a low concentration of coffee drinkers wandering in front of the store, you get a slow rate. If you increase their numbers, the rate of cappuccino production increase. The rate is ‘diffusion limited,’ that is, set by the number of coffee drinkers that wander into the store…until a line forms. At that point, the Barista is working at maximum rate (saturation kinetics) and the rate of cappuccino production is limited by the catalytic rate, how fast the Barista can work. The only way to increase the rate is to add more enzyme…in this case, just open another Starbuck’s 100 meters away).
Let’s consider two types of inhibitors: one that purely inhibits the first step (binding) and one that alters the enzyme so that it is not as good at catalysis (the second step), but otherwise has no effect on binding.
Competitive: Enzyme-Inhibitor (E-I) complex or Enzyme Substrate (E-S)
Inhibitor1Fig 5.
The substrate does not bind the E-I complex and the inhibitor does not bind the E-S complex. Shown here the two are actually binding to the same site. Inhibitor physically blocks access of the substrate to the enzyme. This could be the idiot who gets to the front of the line at Starbuck’s and cannot think of what he wants to order, but just stands there and won’t let others order.

The substrate will have its binding constant (approximately K
M) and the inhibitor with have its binding constant (lets call that KI). Note that the second step governed by k2 in Fig. 3 is NOT affected by the presence of the inhibitor (The Barista works at the same rate…he’s just not able to get working). If the substrate binds, it will be catalyzed at the same rate as it would if the inhibitor were not in solution. It’s just that fewer substrate molecules get to bind.
Since both the inhibitor and the substrate have an “off rate,” I can assume that even in the presence of the inhibitor, substrate will have some opportunity to get access to the enzyme when the inhibitor comes off.
Let’s assume a simple case in which the inhibitor and the substrate have similar binding constants for the enzyme. Then, if I had equal amounts of each, I would expect at any given time that half the enzyme will have substrate bound and half will have inhibitor bound. If I increase the concentration of substrate, I would be able to get more of it to bind (out-compete the inhibitor for the site) and the enzyme would be able to do its job as normal. At a great enough excess substrate concentration, the probability of substrate binding is much higher than that of inhibitor binding; so most enzymes would have substrate bound. I could eventually get the rate up to the V
max. But, the KM becomes much need much more substrate to get the enzyme to work at half the max rate.
So, a competitive inhibitor raises the Km, but does not affect the Vmax.

while the mechanism of competition strictly results in inhibition, the other types of interactions described below could just as easily activate the enzyme, or increase, the rate.

Non-competitive: Lower the rate “k2.”
Another type of inhibitor does not affect binding of substrate. Instead, it alters the rate of the catalytic step. How can it do that? Remember that these are proteins. A small shift tertiary structure caused by the binding of inhibitor could change the ability of the enzyme to catalyze the reaction.
This effect where binding of a regulator (inhibitor or activator) changes the shape of the enzyme and alters its rate are call
“Allosteric effects.” Allosteric just means binding somewhere else and not directly interfering with substrate binding.
In that case, I’d get binding at the same rate. So, K
M would not change. However, since k2 is lower, I have a lower Vmax.
Easy. This would be the girl behind the counter who is really “into” the Barista and is chatting to him non-stop. That slows down his catalytic rate, but doesn’t affect the rate at which customers wander in.
All non-competitive inhibition is allosteric. However, not all allosteric effectors show non-competitive inhibition. Some are even activators.

You may notice that I have more steps for my enzyme than the book has. The substrate has a binding constant, what about the product? Wouldn’t I expect the product to have some binding affinity for the enzyme? And, the product has to leave the binding pocket in order for new substrate to fill it. In effect, the product can act as a competitive inhibitor of the enzyme that produced it. This is the very simplest form of “feedback inhibition,” an important concept we will talk more about. We refer to the combined rates for the steps of catalysis and releasing product as an enzyme’s “turnover rate.” That is, how fast it is available to do its job again.

Much of the time, I don’t want an enzyme to work at full speed. One very important way of regulating an enzyme is feedback inhibition, as in the simple product competitive inhibition mentioned above. Suppose I have a pathway requiring 3 enzyme steps.
Once I have enough D, I don’t want the pathway to be running at full speed. Maybe “A” is also needed for another pathway (not at all unlikely) and once I have enough D, I want to divert A down that other pathway.
One real world example would be if "A" were glucose. Sometimes I want to use my glucose to get energy right now. Sometimes I want to store glucose in glycogen for later use. Which path I choose would depend on changing conditions. A well-evolved system should adjust to needs.
One way to regulate this is if D is an inhibitor of enzyme 1. It may be competitive or non-competitive.

activation is common as well.

This sounds magical, until you remember that we are talking about proteins, composed of helices and sheets arranged in specific tertiary structure. Couldn't the binding of some activator on one face of the protein cause to helices to shift relative to each other? Of course. Couldn't the shift slide a helix out of the way of the active site, allowing better access? Sure.
The possibilities for regulation seem endless, really. And every possibility I've ever thought of and about 100x more that I never thought of can be found operating in nature.