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  • JOANNE STUBBE: We're talking about the purine biosynthetic

  • pathway.

  • Here's the pathway.

  • I told you, in this part of it we were going to go through,

  • so at least you saw what the steps in the pathway are.

  • The key thing is you start out with the ribose 5-phosphate,

  • and then you build up the base a step

  • at a time, which is completely different from pyrimidines,

  • where you make the base, and then stick

  • on the ribose 5-phosphate.

  • And I told you at the very beginning,

  • there were a few interesting steps

  • in this pathway that are universal in almost

  • all metabolic pathways.

  • And one of them we were going over-- two of them

  • we already went over.

  • I'm going to briefly go back over this,

  • but the role of glutamine in the purine and pyrimidine pathway

  • as the source of nitrogen. There were five of these enzymes.

  • That's not an accident.

  • Glutamine is one of the major ways you deliver ammonia

  • into molecules.

  • And purines and pyrimidines both have a lot of nitrogens.

  • The second thing we were talking about,

  • and we had gone through the first few steps here,

  • was the second enzyme in the pathway, where we use ATP,

  • and in this particular pathway, this

  • is the mammalian version of the pathway, which

  • is pretty similar to the bacterial,

  • but there were five different steps that require ATP.

  • This pathway demonstrates how you

  • see ATP use over and over and over and over again.

  • There are defined structures for the binding sites of the ATPs.

  • Once you have these in your brain, it becomes easy.

  • You might not know which one of these mechanisms it is,

  • but after you do a little bit of reading, or bioinformatics,

  • you can immediately tell what the structure of the enzymes

  • actually are.

  • The other thing we talked about already was the role of folate.

  • Those are the three things I want you to get out of this,

  • and we're going to go through the rest of that today,

  • and then, after we finish that, we'll come back

  • to the purinosomes, which is the reason

  • I chose this topic a long time ago,

  • because it speaks to the question of the importance

  • of transient protein-protein interactions in metabolism

  • inside the cell, which has been something that people have been

  • interested in for decades, and this paper in 2008

  • that you read for recitation was very

  • interesting to a lot of people, and we'll come back and talk

  • about that at the end.

  • The first enzyme-- the names are horrible.

  • I gave you the names of all these things.

  • If you look at last year's exam, you

  • will have the purine pathway with the name stuck at the end.

  • I don't expect you to remember this, but we go from PRPP--

  • we've already gone through this step--

  • and the enzyme is PurF--

  • I'm not going to write it out--

  • goes to PRA.

  • The reason I'm writing that again is because a key reason

  • that Bankovic's lab and my lab, many years ago,

  • was focused on this is because of the instability

  • of the intermediates in this pathway.

  • This guy has a half life of 15 seconds at 37 degrees,

  • so this is chemically unstable.

  • This is enzyme 1, and this is the first place

  • we saw glutamine going to glutamate

  • as the source of ammonia.

  • And I wanted to go back and say one more thing about that.

  • Again, there are two enzymes that use glutamine

  • as a source of ammonia.

  • This one is simply, if you look at the pathway displacing

  • pyrophosphate ammonia, you have a nucleophile displacing

  • pyrophosphate which, when complexed to magnesium,

  • is a good leaving group.

  • The idea here is that all of these proteins,

  • and there were five of them, in the purine and pyrimidine

  • pathway, have two domains.

  • Sometimes the domains are separate polypeptides.

  • Often they're linked together.

  • The glutaminase domain is in one of these domains,

  • and the way the chemistry goes, the way

  • the ammonia is going to displace whatever the leaving group is

  • in the second domain, requiring a tunnel that

  • varies from 25 to 40 angstroms to actually mediate

  • ammonia release.

  • PurL is the fourth enzyme in the pathway.

  • Again, here's the glutaminase domain.

  • It's upside down, and here's where the chemistry

  • occurs in the other system.

  • What I wanted to say about that is

  • that all of these enzymes in the active site have a cysteine.

  • All of these enzymes have a cysteine in the active site,

  • and you should go back and look at the PowerPoint,

  • because I'm not going to write this out on the board.

  • You've seen this chemistry now, over and over again,

  • but, in some way, the glutamine is

  • going to be attached covalently with loss of ammonia

  • to a cysteine in the active site.

  • Let me show you what the mechanism of that is.

  • Here is a generic mechanism, but it

  • could be a cysteine protease.

  • These are the same things we've seen over and over again,

  • so this should now be part of your basic vocabulary.

  • So the goal, then--

  • here's our glutamine-- is simply to liberate ammonia.

  • The cysteine needs to be activated somehow

  • for nucleophilic attack.

  • How is that done, normally?

  • With a histamine.

  • This particular enzyme.

  • There are two superfamilies of enzymes that do this.

  • This one doesn't use histamine, but it still

  • needs to be activated.

  • You go through a tetrahedral transition state, which

  • collapses to form an acylated enzyme, and, in the end,

  • you need to hydrolyze this off to give you a glutamic acid.

  • One of the reasons I wanted to go back to this,

  • again, is because, in the Bankovic paper,

  • we talked about, but didn't go through

  • in any detail, the fact that, in that paper,

  • to study whether these purinosomes could assemble

  • and disassemble, they use an inhibitor

  • of the purine pathway, which then should

  • want the enzymes to assemble, because they

  • need to make purines because you've blocked the pathway.

  • And the inhibitor they used is a molecule that looks like this.

  • They used azaserine, but it has another methylene in it.

  • This is DON.

  • And this is a diazoketone.

  • This is a natural product, and it

  • was discovered by Buchanan's lab at MIT,

  • and it was the first diazo compound that people had seen.

  • And it inhibits all--

  • this is something that's important

  • when thinking about what's happening

  • when you're treating cells with it to stop purine metabolism--

  • it inhibits all glutamine-requiring enzymes,

  • because the mechanisms are similar.

  • So the mechanism, if you sit down and think about it,

  • is pretty simple.

  • You have a diazo group, and now the proposal

  • is that this needs to be protonated

  • by the cysteine in the active site.

  • And now you have an N2 to that's dying to leave, N2+,

  • and so you just do an SN2 reaction forming a covalent

  • bond.

  • That's the basis for how azaserine in the Bankovic paper

  • works.

  • There was another way that they block

  • the pathway, which hopefully we'll have time

  • to come back to in the end.

  • So, again, this idea of coming together and going apart-- how

  • do you perturb this?

  • One way they perturbed it was depletion of purines.

  • We discussed that.

  • We didn't really discuss this particular step.

  • The next step in this pathway.

  • Now we have R, which is ribose 5-phosphate.

  • I'm not going to write that out, because every single step now

  • has ribose 5-phosphate as a scaffold.

  • And what we added was glycine.

  • Again, here's the first time that we need to use ATP and Pi.

  • Lots of times, you don't know, when you look at this,

  • whether you're going to transfer pyrophosphate

  • or you're going to phosphorylate,

  • so where you have attack on your ATP.

  • Almost all the enzymes, but not all of them, in the period

  • pathway have ATP going to ADP, so that tells you

  • the attack has to be on the gamma position.

  • This is an ATP grasp superfamily member,

  • and they all go through the same mechanism, which I briefly

  • talked about last time, so I'm not going to write this out

  • again, but basically, you're going

  • to go through a phosphoanhydride, which is then

  • attacked by a nucleophile.

  • We're converting the hydroxyl group of the carboxylic acid

  • into a good leaving group.

  • You've seen this used over and over again

  • over the course of the semester.

  • But over here, this is all written out for you.

  • Here we have glycine.

  • R is CH2NH2.

  • You phosphorylate to form the anhydride.

  • You still need to neutralize this to make it

  • into a good leaving group, which is done in the active site,

  • and then you can have a variety of nucleophiles

  • that could come in and attack to form the covalent linkage.

  • In this case, the nucleophile is not the NH3+.

  • It needs to be converted to the NH2--

  • Sorry.

  • The nucleophile is over here.

  • It's phosphoribosylamine.

  • So it's the NH2 of the phosphoribosylamine that's

  • attacking.

  • Again, to be a nucleophile, it's got to be deprotonated.

  • Hopefully, you all know that at this stage.

  • So what do these enzymes look like?

  • They all look the same.

  • It turns out that if you look at, globally,

  • purine biosynthesis, not just focus on mammalian systems,

  • there are four or five enzymes that

  • actually are ATP grasp superfamily

  • members in the purine pathway.

  • And they all look like this.

  • They have a little domain with a lid,

  • and all the chemistry happens in between,

  • and the lid opens and closes.

  • You can pick these out by bioinformatics.

  • That's the second step in the pathway.

  • And this just shows what all of the products can be,

  • so if you go back and you pull out the pathway,

  • there are ATP grasp superfamily members,

  • and these are the products that are

  • formed by this common type of mechanism

  • through a phosphoanhydride.

  • The next step in the pathway.

  • So now we formed--

  • The next step in this pathway, let's see if I put this.

  • All right.

  • Sorry.

  • I thought I put another copy of this in.

  • The next step in the pathway is we need to formylate.

  • What do we use as formylation?

  • That's why we spent the introductory part

  • of this course talking about folates,

  • which can transfer carbon at three different oxidation

  • levels.

  • What you have here is, and I'm not

  • going to draw the whole thing out,

  • this is the part I told you was the business end.

  • This is N10-formyltetrahydrofolate.

  • Theoretically, this could be either here or here,

  • and chemically they can actually interconvert

  • under certain kinds of conditions.

  • But we know, for all purine pathways

  • that people have looked at, it's always the N10.

  • That's distinct from methylation,

  • where it's always from the N5.

  • I don't know how things evolved, but that's

  • what the results are.

  • How does this happen?

  • Hopefully, you all know this without me

  • having to write this down, but this needs to be a nucleophile.

  • It needs to be deprotonated.

  • You need a base to remove a proton,

  • and then you form a tetrahedral adduct, and then

  • the tetrahedral adduct high energy intermediate collapses,