All right! Final stretch! I’m not even going to bother trying to pretend that I’m super psyched about this. Honestly, if it weren’t for the sweet sound of anime playing in the background (…what? It’s in Japanese, so it’s less distracting than normal stuff! Okay…? [mutter mutter]), I’d probably have lost my mind by now. Still, this subject isn’t altogether unpleasant. In fact, it’s happily specific—we’re going to learn about thymine, uracil and cytosine!
Pyrimidines are the simpler of the nucleotides that we have in our bodies, but that makes it sound like they’re simple molecules. If you’ve seen one on paper, you know this isn’t the case—even uracil, a relatively uncomplicated one, is more complex than we’d like it to be.
Now, we know that our body wants these things around, because they’re ridiculously useful. Nucleotide triphosphates (ATP, CTP, etc.) are used as energy currency for countless biological reactions. RNA nucleotides are used to make RNA. Reduced forms of these (deoxy versions) are used to make DNA.
So, how do we make them? Well, it’s reasonably complicated, as you might expect, but it’s less so for these pyrimidines. Turns out, no matter which one you set out to make, they all go through the same conversion: orotidylate (OMP) to uridylate (UMP) to cytidylate (CMP). In all of these, the stereochemistry of the bond between the sugar and the nitrogenous base is the same: it’s the beta-N-glycosidic bond that we saw way back at the beginning of this semester.
Before we can do anything, though, we have to start by turning ribose-5-Pi into something that will readily assimilate into a nucleotide. This is easier than you’d think, but it requires quite a bit of energy.
See, what happens is that an enzyme called PRP Synthetase uses ATP to convert ribose-5-Pi to 5-phospho-ribosyl-pyrophosphate (PRPP). PRPP is a really reactive species, and it’s more than happy to humor us when we start trying to put it into nucleotides. As you can see from the generation of that PPi at the anomeric carbon, this converts ATP into AMP.
All right, now that we’ve got that starting material, we can start building up our pyrimidine bases. That’s a bit tricky for us, apparently, because we’re not going into gritty details, but we do have the basic gist of things:
Glutamine, bicarbonate and aspartate are gathered together and assembled into orotate. This is a series of four reactions that takes two ATP to accomplish. Bicarbonate and aspartate are used as a carbon source, and aspartate and glutamine provide nitrogen.
Once this occurs, orotate reacts with PRPP to make OMP, spitting out pyrophosphate (which is hydrolyzed into two Pi). This happens using the enzyme Phosphoribosyl Transferase, and it’s unsurprisingly spontaneous.
OMP is then decarboxylated, producing UMP. This is accomplished using the enzyme Orotidylate Decarboxylase, which, surprisingly, isn’t dependent on PLP like most of our decarboxylases are.
Two phosphorylations of UMP are accomplished using ATP as a source of phosphate, producing 2 ADP and UTP. These happen using nucleoside phosphate kinases, both a monophosphate kinase and a diphosphate kinase. These reactions take advantage of the high concentration of ATP in the cell to spontaneously accomplish this transfer.
Finally, the C-4 keto group of UTP is converted into an amine group using glutamine as a nitrogen source (and ATP). The enzyme that does this is called Cytidine Triphosphate Synthase, and it contains a channel that funnels ammonia liberated from glutamine to the activated 4-C carbonyl.
Overall, this synthesis requires six ATP, using a total of seven phosphoanhydride bonds. That’s quite energetic, but really, if you think about it, it’s not too big of a shocker—we took really basic molecules (bicarbonate, aspartate, glutamine and ribose-5-Pi) and built them up into an aromatic ring structure attached to a sugar. If that didn’t require energy, I’d call shenanigans. Wouldn’t you?
Now, like every biosynthetic pathway in our bodies, this is regulated depending on what our bodies need. The very first enzyme in the synthesis of orotate, Carbamoyl Phosphate Synthetase II, responds to allosteric activators and inhibitors. Unsurprisingly, the activator of this pathway is ATP, since ATP is a major requirement of this biosynthesis. Also unsurprisingly, the inhibitor is CTP, since CTP is our final product.
Now, this is the part of the post where I usually go, “Okay, now let’s talk about catabolism!” However, before we do that, we actually have a couple of branch points we can investigate. You see, for some syntheses, our body doesn’t use ribonucleotides—it uses deoxyribonucleotides. If you don’t believe me, ask a genetics professor. They’ll tell you all about a little biomolecule called deoxyribonucleic acid (DNA).
The way we make deoxynucleotide versions of our nucleotides (whether they’re purines or pyrimidines) is pretty straightforward: we use an enzyme called Ribonucleotide Reductase. This guy uses NADPH to reduce nucleoside diphosphates (and only diphosphates) to their deoxynucleoside diphosphate forms by removing the 2′ OH group that distinguishes the two. Put in ADP? Get out dADP. Put in CDP? Get out dCDP. Put in UDP? Get out dUDP.
Wait… we don’t use uracil in our DNA, do we? That’s one of those things they taught us in high school chemistry—uracil is strictly used in RNA.
Well, you’d be right there. We don’t use dUDP to make DNA. However, do you remember which nucleic acid uracil replaces? That’s right, it replaces thymine. I bet your mind won’t be blown when I tell you this: that’s because they’re structurally similar.
In fact, the only difference between uracil and thymine is a methyl group at the fifth carbon. Doesn’t that suggest that we should be able to make one from the other? Well, you’re right, reader! Turns out, the enzyme Thymidylate Synthase exists.
Thymidylate Synthase is an enzyme that turns dUMP (and only dUMP) into dTMP using methylene-THF. (If you’re thinking “tetrahydrofuran,” ‘grats! You’re an organic chemist! However, what we’re actually going for here is N5, N10-methylene-tetrahydrofolate.) This is a conversion driven by high energy charge, and it only occurs with the monophosphate (no dUDP or dUTP).
All right, now we can put it all together! It seems like it should be complicated, but it’s really not that bad. (You should see the slide in my professor’s notes. He told us, “I saved this until after the withdrawl deadline. Now you can’t escape.”) Just follow my lead, okay?
Beginning with our starting materials, we build up UMP (through OMP, remember). UMP can only do one thing to contribute to biosynthesis: be phosphorylated into UDP and UTP. UDP can be converted into dUDP using ribonucleotide reductase, and UTP can be converted into CTP using cytidine triphosphate synthase. (Note that the phosphorylation matters.)
CTP can be dephosphorylated into CDP and then made into dCDP. Both dUDP and dCDP can be phosphorylated back to their triphosphate forms, but dUDP can also be dephosphorylated and turned into dTMP using thymidylate synthetase. dTMP can then be phosphorylated into dUTP.
All right, so starting with just UMP, we’ve made UTP, CTP, dUTP, dCTP, and dTTP. However, what happens if we want to go the other way? What if we want to degrade these suckers?
Well, under catabolic conditions, while synthesis is slowing down, enzymes called nucleotidases are just getting started. These convert nucleotides to nucleosides by hydrolyzing off the phosphate groups. dCMP and CMP become dCytidine and Cytidine, dUMP and UMP become dUridine and Uridine, and dTMP becomes dThymidine.
Next, pyrimidine nucleoside phosphorylase cleaves the beta-N-glycosidic bonds between bases and sugars using inorganic phosphate. This generates cytosine, uracil, thymine, and ribose-1-Pi. Ribose-1-Pi is isomerized to ribose-5-Pi, which is then used either for energy production or nucleotide biosynthesis.
Finally, a deaminase converts cytosine into uracil, which is metabolized into beta-alanine, ammonia and bicarbonate. This is accomplished using a pathway that breaks the aromaticity of the ring before hydrolyzing it. Thymine meets a similar fate, although it is metabolized into beta-aminoisobutyrate instead of beta-alanine.
Well, that’s it, everyone! Biochemistry is over! I’m not sure about you, but I’m getting a little emotional. It’s weird to think that this was my last introductory biochemistry lecture. Oh well, I won’t get too upset about it—I only have to wait until next semester, when the fun that will be advanced biochemistry begins.
In the mean time, enjoy your lives, friends! I may post a little bit on transition metal chemistry before tomorrow, but if I don’t, please enjoy the silence in your Facebook feeds while you can. I probably won’t be gone for too long. ❤
Now, anybody have any good strategies for completing several months’ worth of physics homework in three days? Or perhaps an effective study method for a Chinese test? Anyone?