Respiration and Photosynthesis

Here is a
link to a "mind map" comparing and contrasting the two.


First, Here is a link to Richard Feynman explaining the two slit experiment


Plants are examples of “Autotrophs,” or, organisms that can support themselves and don’t need to eat other things (we are “heterotrophs;” we do eat other things). In particular, plants are “photoautotrophs,” supporting themselves with light.
This is the basic scheme we have been following. Photosynthesis can be broken into two parts, the “photo” part (light reactions” and the “Synthesis part” (Calvin Cycle). The light reactions produce two main things: ATP and NADPH (It’s just like NADH, except it has an extra phosphate). The processes that result in look like the ones we learned about in respiration (except, backward). The ATP synthase works the same way as it does in respiration.
The ATP and reduced nucleotide cofactor (NADPH) are used in the synthesis part (Calvin Cycle) to pull CO2 out of the air and synthesize carbohydrates. In the process, the oxidized NADP+ and ADP are produced…and fed back into the light reactions.

Light Reactions

The general idea is that photons of light hit electrons, exciting them to a higher energy level. Instead of “falling” back down and emitting light, the high energy electrons are passed to other molecules, reducing them, and the electrons are passed down an electron transport chain of proteins or smaller molecules that are reduced, then oxidized by the next player in the chain. In this way, just like in respiration, protons are pumped across the membrane and those drive the ATP synthase. The first recipient in the chain is called “Plastoquinone” and it looks like this:
(That structure in the brackets is repeated 9 times). (The protein complex that gets the electrons next is called “cytochrome b6f”). There are two different photosystems (called PSI and PSII) that can work together. Photosystem I can also work on its own. Each has their own chlorophyl and accessory pigments in the “antenna complex.” In a pattern known as the “Z scheme,” electrons starting in PSII get passed though electron transport to PSI, where light excites it. Another electron RedOx reaction can pass two electrons onto NADP+, generating NADPH, the reduced form. This will be passed to the “synthesis” part, or Calvin Cycle. The Z scheme looks like this:Thylakoid_membrane

Some key points:
  1. In the Z scheme, PSII is excited “first,” (there are many reaction centers all being hit more or less simultaneously. It’s just that to see the “scheme,” we have to imagine things happening in sequence).
  2. Electrons leave PSII. They are replaced by electrons taken from oxygen in water (a rare case of oxygen being oxidized). This generates O2, which is good for us.
  3. At some point PSI also gets hit by a photon of the right energy. That electron can be passed directly to an iron-containing protein called “Ferrodoxin” and eventually used to reduce NADP+. The electrons are replaced by electrons coming down the from the PSII system.
  4. The Z scheme is also called “linear flow” of electrons because they appear to start at one place (PSII) and pass down the chain, eventually leaving the system. You could call it an “open” pathway, since electrons will leave the system at the end, transferred to NADP+ to make the reduced NADPH, and therefore need to be replaced by electrons from oxygen, obtained in PSII.
  5. Photosystem 1 (PSI) can work on its own without PSII. In that case the electrons excited from PSI get passed down the chain to Cytochrome b6f and passed back into a PSII reaction center. When this happens: Oxygen is NOT released and NADPH is not made. In some organisms, the electrons can be taken from some other source and NADPH can be made, but, oxygen is not produced. (So, it is not necessarily true that you need both systems to perform the "synthesis" part.
  6. From an evolutionary point of view, PSI may be older. If that’s the case, photosynthesis was going on for a long time without oxygen being produced. This is still somewhat debated.

The "Synthesis" Part

Basic. There are three phases.

  1. Carbon Fixation: The starting molecule is Ribulose 1-5 bisphosphate, a 5-carbon sugar derivative with a phosphate on each end. CO2 is pulled from the air and attached to one end, creating a very unstable 6-carbon molecule that breaks almost immediately in to two 3-carbon molecules called 3 phosphogycerate. If you’re counting Carbons (and you should be), you started with 5, added 1 CO2 to get 6, then broke that into two 3-carbon molecules.
  2. Reduction: This molecule may be used for some biosynthetic pathways. However, to be really useful, it needs to be reduced from the carboxyl (COO-) form to the aldehyde form (C=O). This goes in two steps involving first ATP, then reduction with NADPH. This yields the very useful glyceraldehyde 3-phosphate (G3P). I’m sure you remember that one from glycolysis.
  3. Regeneration: For it to be a “cycle,” the starting material Ribulose, 1-5 bisphosphate has to be regenerated. This actually has several steps and if you don’t look a little at the details, the math doesn’t seem to work. For that reason, I’ll do a little detail here. What happens is the first two steps are run 3 times, (requiring 3 Ribulose 1-5 bisphosphate molecules), which gets you 6 G3P. One of those can be syphoned off to use for other things such as glucose synthesis. The remaining 5 G3P are rearranged through several enzymatic steps to get you back your 3 Ribulose 1-5 bisphosphate. (Five 3-carbon molecules get you three 5-carbon molecules…15 carbons in each case)
So, running the Calvin cycle 3 times gets you a net gain of one G3P. Run it 3 more times and you can use two of those to make a glucose. That takes more ATP and is essentially the reverse of the first few steps of glycolysis. It takes a total of 6 times round the cycle to make a single glucose.


You don't need to memorize this. It's here for context. Please keep reading. There is more after this.
  1. We start with Ribulose 1-5 bisphosphate (that means 2 phosphates, one on each end of the molecule at positions 1 and 5). Note that ribulose is a ketose, with a carbonyl at position 2. The attack of the CO2 is at position 4, one from the right end.
  2. RuBP-2D-skeletal
  3. The intermediate, 3-keto-2-carboxyarabinitol-1,5-bisphosphate (hope you appreciate that I wrote out the name of the “β-keto intermediate”, Brian), is immediately broken into two 3-phosphoglycerates. These have a phosphate on position 3, a carboxyl group (acid) at position 1. Note that here they are assumed to be deprotonated, as they would be at normal pH.rubismech
  4. Phosphoglycerate can itself be used as starting material for many biosynthetic pathways. Some molecules may get syphoned off for other uses.
  5. ATP is used to add a phosphate to the other end (carbon 1) of 3-phosphoglycerate (3-PG), yielding 1,3 bisphosphoglycerate (you need two ATP because you have two 3-PG). Note that the carboxyl end (left side) now goes COOPO3-. In ATP hydrolysis, ADP would be left with the extra oxygen and the Pi picks up its fourth O from water. Here, the transferred Phosphate gains its fourth Oxygen from the carboxyl.
  6. NADPH is the reducing agent for the next step, which removes the phosphate. Note that if you now remove the Pi, the oxygen that was the end of the carboxyl is removed, leaving just the carbonyl. So, it’s now an aldehyde (terminal carbonyl) and is Glyceraldehyde 3-phosphate. It can be used in biosynthetic pathways too. The H from NADPH is now at the end of the molecule, where the O used to be.
  7. Once you build up enough G3P, you can syphon one off for Glucose synthesis (note that this particular version of the cycle implies that it’s the 3 phosphoglycerate that is syphoned off. This is a bit of a controversy...and may just be a point of view. Let’s stick with the G3P as being the main intermediate used to build glucose.
  8. I’m not going to go into any more detail on the regeneration process than I did above in phase 3.

CAM and C4 Plants

The problem of Oxygen

We discovered that Photosystem II has such an extremely high RedOx potential that it can oxidize Oxygen, removing it from water and releasing it as molecular O
2. That’s good for us, but creates quite a few problems. The main problem as far as we are concerned here is that RuBisCO has a very odd property: it can carry out a very unproductive reaction called “photorespiration.” In this reaction, instead of binding CO2 and adding that to ribulose-bisphosphate (RuBP), it binds O2 and adds that to RuBP.
RuBisCO binds CO
2 preferentially. But, if there is a lot of O2 around photorespiration happens.
The exact biochemistry is not that important here. There are a whole series of reactions that take place and the net result is that CO2 is released (that’s why it’s called “respiration;” oxygen is consumed and CO
2 is released). But, unlike cellular respiration, ATP is consumed in the process. Thus, it is considered a wasteful process.

There is some debate how much of a problem this is for plants. Photorespiration may be helpful to some plants under some circumstances. However, it
is clear that other plants have evolved specific strategies to minimize it.
Plants that have no strategy to prevent photorespiration are called C3 plants (because the product of carbon fixation are two, 3-carbon molecules…3 phosphoglycerate). They do photosynthesis the way I have taught it so far. They include most of our agriculturally important plants, such as wheat, rice, soybean, potato, fruit trees and nut trees and others. They tend to do well in cool, moist climates, but not at all well in hot, dry climates. In such climates, the plants will keep their stoma closed to conserve water. Gradually, this will deplete the CO
2 and build up O2,) leading to photorespiration).

The Biochemistry

Plants that live in hot, dry climates have evolved mechanisms to prevent photorespiration. Neither evolution nor modern protein engineers have managed to produce a RuBisCo version that does not carry out photorespiration.
So, plants in hot, dry climates have taken a different approach: keep oxygen production away from RuBisCo and keep CO
2 as high as possible around it.
One such type of plants is called C4 plants and the other CAM plants. Both these strategies involve keeping the concentration of CO
2 relatively high when the Calvin Cycle is running. While there is some variation on these themes, the biochemistry is similar in both cases. I will present the simplest variant:
Both fix CO2 onto phosphoenolpyruvate (PEP) forming the 4-carbon molecule Oxaloacetate. That is converted to Malate by the enzymes indicated.

CO2 can be released from Malate, generating pyruvate. Remember “Pyruvate Kinase” from glycolysis? It will take pyruvate and add phosphate from ATP to regenerate PEP so the plant can do this again.

C4 Plants: Spatial separation.

These include a few important agricultural plants, such as corn and sugar cane as well as some grasses. “Crabgrass” is a C4 plant, which is why it takes over your lawn, especially in dry weather.
When grown in the same environment, at 30°C, C3 grasses lose approximately 833 molecules of water per CO
2 molecule that is fixed, whereas C4 grasses lose only 277 water molecules per CO2 molecule fixed. This increased water use efficiency of C4 grasses means that soil moisture is conserved, allowing them to grow better in arid environments.
Thus, the grass we like requires roughly
THREE TIMES the water as crabgrass. I still hate crabgrass.

C4 plants separate oxygen production from the Calvin cycle in two different sets of cells, known as the “mesophyll” and “bundle-sheath” cells, respectively. The set up looks like this:
Pasted Graphic 6
This image is used without permission from this website:

They transport the malate made above into the bundle-sheath cells and released the CO
2 in the chloroplast of those cells. This keeps the concentration of CO2 high in those cells and minimizes photorespiration. Another depiction of the overall scheme is below:

CAM Plants: Temporal separation

CAM stands for Crassulacean acid metabolism. There is no chemical named “Crassulacean acid.” The name comes from the family of plants that use this approach,
Crassulaceae, and the fact that the chemicals oxaloacetate and malate are organic acids.
CAM plants include succulents. The only agriculturally important plants in this category are pineapple and agave.
They keep their stomata closed during the day, minimizing water loss. Then, at night, when temperatures are generally cooler, they open stomata and allow gas exchange. They save the Malate generated in the pathway above in the vacuole, then use it during the day to keep CO
2 concentrations high. This image is from wikipedia and yes, the labels are in German.

Side point:

PBS ran a documentary by Ken Burns not long ago on the “Dust Bowl,” in case any of you have trouble falling asleep, you can look it up. However, there is a good point to be made connecting this element of history to biology. The native grasses on the great southern plains, such as buffalo grass, Are C4 plants. These have typically deep roots and use less than ⅓ the water for each glucose generated than the shallow-rooted C3 plants, such as wheat, that we planted there in their place.


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.
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