Respiration Continued

Some details on where the high-energy electrons come from.
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Intro Respiration

Big Picture:
You should review this figure on mitochondria:
https://upload.wikimedia.org/wikipedia/commons/b/b5/Mitochondrion_mini.svg
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


  1. Enter the ATP synthase:
  2. 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.”
  3. 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.

  1. 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.
  2. 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.
  3. 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.”
CoQ
  1. You will see these structures over and over. They will always be doing similar tasks.

Oxidative phosphorylation.

  1. 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.
  2. I stole the figure below from a great text book called “Molecular Cell Biology.”

ETS2
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
  1. Most well characterized cytochromes.Cytochrome_C 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.
  2. 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.
HemeHemeSpacefill
  1. 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.
  2. 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.
  3. 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.

Cofactors

Small-ish molecules that are bound to a protein and required for it to complete it's function. Read More…

Enolase detail

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.
Enolase1

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:
MetalBinding

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.
ActiveSite


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

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.
Substrate:
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.

Fig.3EnzymeSteps
“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).
Inhibitors
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 higher...you 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.

Note:
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.

Regulation
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.
Pathway
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.

Allosteric
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.