Nicotinamide Adenine Dinucleotides

Greetings, Earthlings! It is I, Junebls Protonbro, here to bring you another installment of biochemistry!

I know I promised to talk about ATP Synthase, but once I write about that, I’ll probably throw in the biochemistry towel and say, “Eh, that’s enough for one lifetime, right?” Plus, Sapphire and I both agree that, no matter how much we read this stuff, it doesn’t sink in. Thus, I take a detour from the amazing machinery of aerobic respiration to something perhaps more important to the whole setup: NADH!

When we talk about cellular respiration, we talk a lot about an unassuming little reducing agent called “NAD+.” Like I said in my post on the ETC, this is a really nice thing to have around, because once you force electrons down its throat, it’s more than happy to give them up to complexes that can use their energy.

However, as I also said when talking about the ETC, we can’t keep just a ton of its reduced form, NADH, around. After all, NAD+ is what we use to pick up electrons in all of those reactions that exist pretty much entirely for that purpose. The supply of this stuff is pretty limited (about 1 mM in any given cell), and the reduced species is even less concentrated; cells keep the ratio of NAD+ to NADH at about 3:1 at any given time. (This is referred to as a positive redox state.)

Okay, that’s all well and good, but we still haven’t talked at all about what NAD+/NADH actually is. In biology classes, it’s often treated as a black box of electron transport. However, because we’re aspiring biochemists (right?), we get to actually learn what makes it tick.

As you’ve gathered from the title of this post, NAD+ stands for Nicotinamide Adenine Dinucleotide. It’s a molecule that contains two nucleotide monophosphates connected to each other through a phosphoanhydride bond. One of the nucleotide monophosphates is AMP (ATP without two phosphates, remember), and the other is nicotinamide mononucleotide (NMN). The base of NMN is nicotinamide, which is a carboxyamide substituded pyridine ring. The whole setup looks like this:

363px-NAD+_phys.svgNow, you’re probably looking at this structure and going, “Itty bitty positive charge? Itty bitty electron-acceptor. I don’t believe it!” Well, habeeb it! When the substituted pyridine ring picks up a hydride at the para position, the whole thing is reduced to NADH, and that lil’ charge goes away. Of course, the aromaticity in the pyridine ring goes away, too, which, I assume, is why it’s so keen to give that hydride up again. Here’s an image:

800px-NAD_oxidation_reduction.svg(The net reaction at the bottom has the hydride adding as a proton and two electrons, but keep in mind that it’s a hydride. K’?)

Now, let’s take a detour really quick to talk about Coenzyme A. (I realize this is a big diversion, but it’s how my notes are laid out, and it’ll be important in a minute.) This is a really important cofactor in your body that’s used to activate things with carboxylic acids. It’s made up of pantothenate (Vitamin B5), AMP, and a terminal thiol group. This thiol group is important, because when it reacts with carboxylates (such as acetate), it makes a thiolester that is resonance limiting and, thus, high energy. Here’s what it looks like:

800px-Coenzym_A.svgKeep in mind that the important bit is that -SH at the end. Waaaaay at the end. If you don’t think that’s funny, you and God clearly don’t have the same sense of humor.

All right, now, let’s go back to our NADH. Like I said, our cells don’t like to keep a lot of the reduced species, NADH, around, since the oxidized form, NAD+, is what tons of its enzymes use to pick up electrons. So, what does our body do when it starts to run out of NAD+? Well, it uses up the NADH, of course!

(You’re seeing, now, why we talked about Coenzyme A, right?)

All of the reactions that consume NADH are similar in that they all use the same kinds of enzymes: oxidoreductases. That makes sense, after all—any reaction involving NADH pretty much is a redox reaction by definition. There are several cellular pathways that use these enzymes to take electrons from NADH and put them on other molecules, but they fall into two basic categories: aerobic metabolisms and anaerobic metabolisms.

The aerobic pathway is one that we’ve already talked about at length. NADH is first made by conversion of glucose to pyruvate, pyruvate to Acetyl-CoA, and Acetyl-CoA to CO2. Then, the NADH donates its electrons to the ETC, reducing oxygen to water and regenerating NAD+.

This is really the ideal situation for our cells, because it kills two birds with one stone: you restore the proper redox state in your cells, and you get a heck ton of energy in the process.

However, our cells don’t always have proper oxygen. If you don’t believe me, ask your muscles the next time you decide to binge-excercise by spending a million minutes on the rec center’s rowing machine. Instead, they do a really weird thing: they reduce pyruvate using NADH, generating NAD+ and lactate. This is called homolactic fermentation, and it’s an anaerobic process.

Yeast and bacterial cells also do something called alcohol fermentation—this is a process that involves decarboxylating pyruvate to acetaldehyde, then reducing it. This generates NAD+ and ethanol. (Ever heard of “fermenting” alcoholic beverages? Yep, this is what you’re talking about.)

In the case of aerobic respiration, the replenishment of NAD+ is a little more complicated than simply using NADH in the ETC. That’s because our cells, as smart as they pretend to be, are missing something relatively simple: an NADH transporter.

You see, glycolysis occurs in the cytosol, which means that pyruvate is generated in the cytosol. Then a transporter protein moves it into the mitochondrion, where it’s first oxidized and decarboxylated into Acetyl-CoA using the Pyruvate Dehydrogenase Complex, and then pushed through the citric acid cycle. This generates NADH, which is used in the ETC to regenerate NAD+ in the mitochondrial matrix. However, because our cells are genuises, there’s no transporter to move this out into the cytosol, where it’s also needed.

To get around this, our cells employ a hilariously convoluted system called the Malate-Aspartate Shuttle. (It’s not that complicated, but think about it. Just think about it.) In the cytosol, NADH is generated using glycolysis. Then, an enzyme called malate dehydrogenase reduces oxaloacetate into malate using the cytosolic NADH. A transporter protein moves the malate into the mitochondrion, where it’s oxidized to oxaloacetate in the last reaction of the citric acid cycle. Then, an amino group from glutamate transfers to oxaloacetate to make aspartate, which is shuttled out into the cytosol. This gives its amino group to alpha-ketoglutarate and becomes oxaloacetate again.

Lactate fermentation is a bit of a different beast. When cells are low on oxygen, they still need to regenerate NAD+ so that they can keep glycolysis going. To do this, they tack an extra step onto the end of glycolysis. After pyruvate is produced, an enzyme called lactate dehydrogenase reduces it to lactate using NADH (which is produced in glycolysis). The net effect is that the cell still makes ATP, but it doesn’t gain or lose any net NADH. Additionally, instead of producing pyruvate, which is used to carry out additional oxidative steps in aerobic respiration, it makes lactate.

Now, unlike what my introductory bio class would have us believe, lactate is actually a pretty useful little molecule. One way that it can be used is in the Cori cycle.

Essentially, here’s what happens: when your muscles run out of oxygen with which to do aerobic respiration, they start making lactate using homolactic fermentation. Lactate is released into the blood (where it’s normally kept at 1 mM), then taken up by the liver. In liver cells, lactate dehydrogenase is used to oxidize lactate to pyruvate, and then gluconeogenesis is used to convert the pyruvate to glucose. This glucose is released into the blood, where cells like those overworked muscle cells can take it up again.

Finally, there’s alcohol fermentation, which is carried out in bacteria and yeast anaerobically. This takes glucose and converts it into ethanol, producing two ATP and two CO2 in the process. This is accomplished by first irreversibly decarboxylating pyruvate to acetaldehyde using pyruvate decarboxylase (which uses a cofactor called TTP). Then, alcohol dehydrogenase reduces acetaldehyde to ethanol using NADH, replenishing NAD+.

Of course, our liver cells have to figure out how to reverse this when we consume ethanol. First, they use alcohol dehydrogenase to convert ethanol into acetaldehyde using NAD+. Acetaldehyde is then oxidized into acetate using acetaldehyde dehydrogenase. Finally, Acetyl-CoA synthetase activates the acetate to Acetyl-CoA using ATP and CoASH. Instead of using this Acetyl-CoA to make ATP, liver cells funnel it off to make triacylglycerides. Thus, when you drink alcohol, it gets converted directly to fat in your liver.

This also means that, if you take in too much ethanol, NAD+ levels in your liver drop. That means your liver can’t turn lactate into glucose, and you end up with hypoglycemia and lactic acidosis, especially if you don’t eat anything with the alcohol.

Well, there you have it! Is that more than you ever wanted to know about the cycling of NAD+ and NADH in your body? I know it sure is for me. However, it is a pretty important enterprise. After all, without NAD+, we wouldn’t be able to capture electrons from our food, and that whole electron transport chain would look pretty stupid. You know what else the ETC would be stupid-looking without? A little thing called ATP synthase.

Questions? Comments? Put ’em down there!


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