Ohio, munkee-koon! I know I said I’d throw in the biochemistry towel after my post on ATP biosynthesis, but turns out I lied. I was going to just study this on my own, but I seem to retain information a lot better when I write about it here (which, I guess, is the whole point of this blog), so here we are again!
(I know what you’re thinking: “It’s always such a pleasure…“)
In the posts that I’ve written over the past week or so, I’ve done a lot of talking about redox reactions that involve NADH and FADH2. The point of those reactions was rather straightforward: get electrons from food and make energy. As we established, our bodies are pretty darn good at it too—aerobic respiration is about half as efficient as just burning glucose into oblivion, and heaven knows it’s better on our insides.
However, our cells need reducing power for more than just energy manufacturing. For red blood cells, having a strong reducing agent around can make the difference between another productive day of life and a gruesome death by free-radical. For cells that carry out anabolism (such as liver cells), it’s utilized in building up all of those complicated molecules that make entropy throw us dirty looks when we’re not looking.
So, how do we get all that reducing power? Well, sure, we make NADH when we’re tearing glucose apart, but, as we’ve seen, that’s used in manufacturing all of the energy that our cells need. Happily, there’s another important player in the redox game, one we haven’t mentioned yet. It’s the cousin of NADH, NADPH.
You may recognize this lil’ guy from photosynthesis, but don’t be fooled by those introductory bio textbooks—the “P” does not, in fact, stand for “plant.” NADPH is like NADH, but it has a phosphate group attached to it. Although this doesn’t change its ability to be a reducing agent, it changes the enzymes that recognize it. Thus, it can be treated differently than NADH, and is, in turn, used in different ways—to make fatty acids (triglycerides), cholesterol (hormones), and deoxynucleotides (DNA), for example.
In case you’re curious, here’s what this little guy looks like (in its reduced form):
As you can see, it’s just a phosphorylated NADH. However, that phosphate makes all the difference—it completely alters which enzymes recognize it, and therefore which enzymes use it.
All right, time for me to ask the obvious question: where do we get NADPH? Do our bodies place an order at Sigma-Aldrich?
Of course not. We are our own chemical manufacturing company. Because biology is apparently quite frugal, we make NADPH using a pathway that starts and ends with useful molecules. This pathway, which oxidizes a hexose-Pi into a pentose-Pi, is called the pentose phosphate pathway.
This pathway has two parts: the oxidative part and the non-oxidative part. Here’s the net reaction for the oxidative part, wherein NADPH is generated:
1 Glucose-6-Pi + 2 NADP+ + 1 H2O —> 1 Ribulose-5-Pi + 2 NADPH + CO2 + 2 H+
As you’ve already noticed, we’re starting with our cells’ favorite starting material: glucose. (Well, glucose-6-phosphate, but glucose immediately gets phosphorylated when it enters your cells, so same thing.) From that (an aldose), we’re making a molecule of ribulose-5-phosphate (a ketose) and a molecule of CO2, and in the process, we’re generating NADPH.
It’s important to note here that, because glucose-6-Pi is also the starting material in glycolysis (essentially), our cells have to choose which of these pathways to prioritize. Although it doesn’t turn out to be a terrible sacrifice either way, the first enzyme of this metabolism is controlled by cytosolic concentrations of NADPH/NADP+. If this is lower than normal (100:1), cells activate the first enzyme in this pathway, shunting glucose-6-Pi off so that it can make NADPH. This also very neatly ensures that there’s enough ribose-5-Pi to produce nucleotides, but we’ll get to that in a second.
Now, I know I promised that the products of this metabolism were useful. However, if you’re not in my biochem class, you’re probably looking at the ribulose-5-Pi and going, “Da heq is that?” Well, don’t worry. It’ll become more familiar-looking a little later.
All right, now, let’s get down to business! It’s time to figure out how this pathway works, one step at a time.
Step 1: Oxidation of Glucose-6-Pi into 6-Phosphoglucono-δ-lactone
In the first dedicated step of the pentose phosphate pathway, glucose-6-phosphate is oxidized into 6-phosphoglucono-δ-lactone by the enzyme Glucose-6-Phosphate Dehydrogenase. The hydride lost from this reaction is transferred to NADP+, producing our first molecule of NADH. This reaction, like every reaction in this portion of the pathway, is irreversible. Here’s a picture of what’s going on:
In this step, the lactone ring is opened again by an enzyme called lactonase. Lactonase, a hydrolase, uses water to irreversibly open 6-phosphoglucono-δ-lactone into 6-phosphogluconate. Here’s what this looks like:
Finally, an enzyme called 6-Phosphogluconate Dehydrogenase oxidizes the third carbon of 6-phosphogluconate, transferring the the electrons that it gets in the process to NADP+. This initially produces a beta-keto acid intermediate. However, beta-keto acids are unstable, and they spontaneously decarboxylate. In this case, it decarboxylates to ribulose-5-phosphate and carbon dioxide. Here’s a picture:
Well, I promised recognizable products, didn’t I? To get those, we bring in the non-oxidative part of the pathway. This four step part of the PPP is remarkable in that it’s entirely at equilibrium; the directions of the reactions depend entirely on the relative concentrations of reactants and products.
Four types of enzymes are key to this part of the pathway: epimerases, isomerases, transketolases, and transaldolases. Epimerases do what you’d expect—they change a sugar into its epimer. Isomerases change one isomer into the other.
Transketolases and transaldolases are both transferases, but they’re a special breed of transferases. Transketolases cleave the first two carbons from a ketose and transfer them to an aldose, turning an aldose into a ketose lengthened by two carbons and a ketose into an aldose shortened by two carbons. Transketolases are dependent on TPP, from vitamin B1. On the other hand, transaldolases cleave the first three carbons from a ketose and transfer them to an aldose, making a ketose into an aldose with three fewer carbons and an aldose into a ketose with three more carbons. These use a particular lysine residue to react with the keto group in the reactant molecule.
All four of these reactions take place in the nonoxidative part of the pentose phosphate pathway, which is really just a beautiful mess of equilibria. First off, ribulose-5-phosphate is turned into two of its isomers, xylulose-5-Pi (a ketose epimer) and ribose-5-Pi (an aldose isomer) using ribulose-5-Pi epimerase (Fe metalloprotein) and ribose-5-Pi isomerase, respectively. Here’s what that looks like:
After this takes place, a transketolase transfers the first two carbons from xylulose-5-Pi (a ketose) to ribose-5-Pi (an aldose), making glyceraldehyde-3-Pi (an aldose) and sedoheptulose-7-Pi (a ketose). Here’s what that transfer looks like:
Now that we’ve generated those intermediates, a transaldolase takes three carbons from sedoheptulose-7-Pi and transfers them to glyceraldehyde-3-Pi, making fructose-6-Pi and erythrose-4-Pi.Now you’re beginning to see those “recognizable intermediates” that I promised: we’re making fructose-6-Pi! That’s really useful, because it can either be converted into energy (by glycolysis) or back into glucose (by gluconeogenesis).
Finally, we use a transketolase to do one more two-carbon transfer to the erythrose-4-Pi from a xylulose-5-Pi that we made in the beginning. This generates fructose-6-Pi and glyceraldehyde-3-Pi, which are both intermediates in glycolysis and gluconeogenesis. Observe:
It’s even cooler if you look at it this way: if the cell just needs the NADPH (doesn’t need the ribose-5-Pi to make nucleotides), running this pathway doesn’t cost it anything extra on the way to making energy. Glucose-6-Pi, the first intermediate in glycolysis, goes through the PPP and makes NADPH. The PPP then produces fructose-6-Pi and glyceraldehyde-3-Pi, which get funneled back into glycolysis to produce energy.
It gets even more hardcore if the cell doesn’t need ATP. If the cell running this pathway isn’t interested in generating energy in the process, it sets up a spiral of NADPH production using gluconeogenesis. Glucose-6-Pi is pushed through the PPP, producing glycolytic intermediates that could be used to make energy (like they were above). Instead, the cell takes those and uses gluconeogenesis to make them back into glucose-6-Pi. This glucose-6-Pi can then be put through the pentose phosphate pathway again.
Phew! All right, everybody! That’s it for this test’s biochemistry! Thanks for sticking with me (if you have been sticking with me! I’ve got some hits on my little bar up top, so thank you 😉 ). I’m going to go study the minutia of the citric acid cycle’s enzymes now. Until I return with some transition metal chemistry, stay cool, bros!
Questions? Comments? Put them down there. It would make my day. 5real.