Why is GTP, rather than ATP, used in nuclear transport?

Why is GTP, rather than ATP, used in nuclear transport?

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Why is GTP used for nuclear transport and not ATP, given that ATP hydrolysis is used to drive most cellular energonic reactions?


Although this question appears to be either unclear or unanswerable (or both), and shows no evidence of research on the poster's part, I have chosen to address one aspect of it to emphasize a general concept. This, despite the fact that my knowledge of nuclear transport processes is minimal.

Brief Answer

GTP, rather than ATP, is used for nuclear-cytoplasmic transport via importins and exportins, because this process involves one of a large family of 'G proteins' that - as their name implies - operate through the use of GTP. Whereas ATP is generally used to drive endergonic molecular reactions, the hydrolysis of GTP in G-protein cycles can be thought as a device to terminate a molecular process. Whether G proteins adopted GTP rather than ATP merely by chance or for a biological reason is a matter of speculation.

NTP hydrolysis - for fun or profit?

It would appear that the question relates to protein-transporting 'importins and exportins', which I learn from the Wikipedia article on nuclear transport are regulated by the small G protein, Ran. When complexed to GTP, Ran facilitates the export of proteins by exportins, a process that is terminated by hydrolysis of GTP to GDP. The GDP-bound form facilitates the import of proteins by importins, a process that is terminated by GTP displacing the GDP.

So if we consider the role of the hydrolysis of the nucleotide triphosphate - here GTP - it is clearly not to supply thermodynamic energy for an endergonic process such as muscle contraction, light generation or a chemical reaction with a +ve ΔG:

Rather, it is to produce a conformational change in a protein which allows the termination of a process. This termination is generally through the GDP-bound form of the G protein (grey) interacting with some other protein (black):

This diagram is deliberately simplistic so that the general features of G-protein action can be seen. However, I think it illustrates the same point made in Berg et al. Section 15.6. Other examples are signal transduction of hormone action via cyclic AMP and binding of aminoacyl-tRNA to the ribosome.

Why do G proteins use GTP?

This 'choice' of GTP in the original ancestral G protein could have been mere chance, that was subsequently reflected in other G proteins that arose through gene duplication and divergence. That would imply that, at least, it was not disadvantageous. Or as the oldest G proteins are perhaps those involved in protein biosynthesis - a process still catalysed by a ribozyme - the choice of GTP might have reflected an RNA base-pairing interaction in the 'RNA world'.

Alternatively one might suggest a biological rationale for separating two types of energy-requiring processes - perhaps by regulation of the enzyme that catalyses the conversion of ATP to GTP, dinucleoside-phosphate kinase. However I am not aware of any evidence to support this idea.

Hydrolysis of GTP in other contexts

As mentioned in a comment by @Roland, there are instances where GTP is used to drive endergonic reactions (phosphoenolpyruvate carboxykinase) in a manner analogous to the use of ATP, but this is quite different from hydrolysis of GTP in G proteins. I do not believe that any convincing rationale for this has been propounded, nor for the similar exceptional use of UTP in glycogen synthesis and CTP in phospholipid synthesis. This question has been discussed in another post.


NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science 2002.

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Part 2: GTPase Reactions and Diseases

00:00:00.14 Hello. My name is Fred Wittinghofer.
00:00:02.18 I'm an emeritus group leader at the Max Planck Institute for Molecular Physiology in Dortmund, Germany.
00:00:11.08 And this is my second seminar
00:00:14.01 and in the first one I introduced you to the molecular switches called GTP binding proteins or G proteins.
00:00:21.15 And in this second part I will more concentrate on a particular aspect of our research which is
00:00:28.20 dealing with the mechanism of GTPase reactions and how they lead to a number of different diseases.
00:00:34.09 And I will obviously focus mostly on Ras-like proteins that I talked about in my first seminar.
00:00:42.23 So, again, just briefly, the mechanistic cycle for these proteins:
00:00:49.25 They come in two flavors these proteins the GDP-bound and GTP-bound state.
00:00:55.13 They have nucleotide bound very tightly.
00:00:58.03 They need a guanine nucleotide exchange factor to release GDP for GTP
00:01:03.23 because in the cell, there is more GTP than GDP.
00:01:06.11 That's why the protein become loaded with GTP once GDP is off.
00:01:10.06 They have a downstream affect in the GTP bound form interacting with some partner proteins.
00:01:15.10 And in order for the switch to be switched off again, you have the GTPase reaction
00:01:20.27 whereby the protein splits GTP into GDP and Pi.
00:01:24.29 And this reaction is very slow and is catalyzed by proteins called GTPase Activating Proteins
00:01:31.06 which will obviously be the major thing that we will be talking about.
00:01:36.24 So what we're talking about, really, is a really basic biochemical reaction
00:01:43.23 namely, the hydrolysis of phosphoanhydrides and it's similar to the hydrolysis of phosphoesters.
00:01:53.08 For example, when you have phosphorylated protiens which are phosphorylated on the threonine, serine or tyrosine,
00:01:57.24 you have a similar nucleophillic attack on the phosphate by water.
00:02:05.04 And obviously, people have been talking about this reaction a lot because it is a very
00:02:12.15 slow reaction for the reason that the phosphates are highly negatively charged
00:02:19.16 and the approach of a nucleophile, for example a water which is partially negatively charged also,
00:02:25.06 is very, very disfavored. And that's why this reaction is normally very slow.
00:02:30.25 So even though the reaction delivers energy,
00:02:33.17 it is very slow because you have to overcome the very high activation energy
00:02:38.16 which depends on what I just told you about--the negative charges.
00:02:42.04 And the higher the activation energy, you might know from your biophysical courses,
00:02:46.13 that the higher the activation energy, the slower the reaction,
00:02:51.00 because the reaction rate is directly proportional to the activation energy.
00:02:54.18 So, nature, then, uses enzymes to lower the transition state energy
00:03:00.27 and thereby make the reaction faster.
00:03:03.15 And there's an interesting article that I bring to the attention of my students all the time
00:03:07.21 from Francis Westheimer who wrote an article many years ago:
00:03:12.01 Why Nature Chooses Phosphates.
00:03:13.25 Because chemists never use phosphate as a leaving group
00:03:17.13 but in biology it is a very frequent leaving group
00:03:21.16 So, because of just this purpose here, because of just what I showed here
00:03:26.17 the phosphoanhydride bond or the phosphoester bond is kinetically stable
00:03:32.06 in other words you can have your ATP or GTP in water and it stays forever or hydrolyzes very slowly.
00:03:39.22 But thermodynamically it's principally unstable.
00:03:43.00 If you use an enzyme, it lowers the activation energy--you can make this reaction very fast.
00:03:47.14 So that's an interesting article that I would recommend you to read.
00:03:50.27 For example, that's why DNA is stable and that's why ATP is such a wonderful source of energy
00:03:57.12 because it, in principle, delivers energy but is stable in aqueous solution.
00:04:03.28 So let's start then with first of all Ras and its GAP-mediated GTPase reaction because
00:04:10.25 that is really the paradigm for many of the things that have been developed afterwards.
00:04:15.24 So this is the molecular Ras. Its a ribbon representation of the structure
00:04:21.29 of the G domain of Ras.
00:04:24.16 You see its heart shaped because.
00:04:27.17 obviously we like it very much and we solved the structure in Hiedelberg.
00:04:32.00 The city of Hiedelberg's advertisement, Ich hab mein Herz in Hiedelberg verloren
00:04:36.04 which means, I lost my heart in Hiedelberg.
00:04:37.27 But the most important reason it's heart shaped is because
00:04:41.13 people call it the beating heart of signal transduction.
00:04:44.06 And so its one of the most important molecules that regulates
00:04:48.14 important signal transduction processes like growth, differentiation and sometimes even apoptosis.
00:04:54.17 And you probably know a little bit about the signal transduction process because
00:04:59.07 every text book has this version, one of the paradigms of signal transduction chain
00:05:04.01 whereby, for example, a growth factor binds to the cell membrane
00:05:08.27 and binds to its growth factor receptor RTK (receptor tyrosine kinase)
00:05:14.22 whereby this becomes phosphorylated. It recruits the exchange factor SoS
00:05:19.04 which then activates Ras to the GDP bound form.
00:05:22.16 And then, now, Ras interacts with the downstream component which is Raf kinase
00:05:26.15 which is the starting kinase for what is called the MAP kinase module.
00:05:30.24 There, one kinase activates the next downstream kinase which is called MAP kinase kinase kinase.
00:05:36.29 MAP kinase kinase kinase activates MAP kinase kinase and that activate MAP kinase
00:05:40.28 which then goes into the nucleus and activates transcription.
00:05:45.03 So this is a very simplified version of what Ras is actually doing.
00:05:48.16 and this is the first one to be discovered (the first signal transduction).
00:05:54.15 But, now it becomes more and more complicated.
00:05:57.26 This is still a very simplified version but it shows you already
00:06:00.27 the major thing about signal transduction via Ras in that many upstream components come and activate Ras.
00:06:09.18 It's either tyrosine kinase receptors or G-protein coupled receptors
00:06:13.09 or T-cell receptors. All of them can activate Ras.
00:06:16.08 And then Ras can activate, downstream, many components, not just Raf kinase
00:06:20.18 but also a molecule called Ral GEF, PI(3) kinase, PLC epsilon and others.
00:06:26.22 And they mediate a number of signal transduction reactions
00:06:32.12 which somehow are integrated somewhere, let's say in nucleus,
00:06:38.00 by a transcription factor and initiate, when the threshold is right, when the number of reaction is right,
00:06:46.15 it initiates a cellular response which can be proliferation or differentiation or whatever.
00:06:52.03 And I will not deal with any aspect of this because I want to concentrate on the switch-off reaction.
00:06:57.28 So the switch-off reaction is again the scheme you have seen, now, many times
00:07:03.25 but what happens in Ras is also an oncogene
00:07:06.26 an oncogene that has two types of mutations, either the glycine 12 mutated to any other amino acids
00:07:13.10 or glutamine 61 mutated to any other amino acid.
00:07:16.21 And what this does biochemically is, it blocks the GAP-mediated GTPase reaction.
00:07:23.21 So you can imagine what happens, you have blocked the return to the inactive state
00:07:29.13 and that's why you now accumulate Ras in the GTP bound form.
00:07:33.09 You don't need any of the upstream signaling anymore because Ras is already in the GTP bound state.
00:07:39.03 And now it has an effect that is not regulated anymore and that's why it leads to cancer.
00:07:44.14 So these simple mutations. and that's why, obviously, as structural biochemists
00:07:49.23 it is a very interesting project to ask the question:
00:07:52.00 How can it be that a single point mutation has such dramatic consequences?
00:07:58.19 And its also. obviously since Ras is the most frequent oncogene.
00:08:02.28 About 25% of the people that come to the clinic diagnosed with a tumor,
00:08:09.07 they have a Ras mutation, one of the ones I showed you. At least these are the most frequent ones.
00:08:15.00 So obviously every drug company also are working on trying to inhibit the Ras pathway
00:08:20.29 and Ras signaling as a way of treating Ras-mediated cancers.
00:08:25.28 And there are many approaches that one can think of.
00:08:30.17 I just indicated a few here. For example, you could think of Ras. is farnesylated
00:08:36.25 at the C-terminal cysteine and there's an enzyme called farnesyl transferase that mediates that reaction.
00:08:44.18 There are farnesyl transferase inhibitors that are just in the clinic being tested for their efficacy.
00:08:50.00 But you could also think of maybe inhibiting the interaction with downstream effectors.
00:08:54.15 This is, for example, a structure we determined: the complex between Ras and Raf kinase (or part of Raf kinase).
00:09:00.10 Or you can even think of. and that's what we're working on still.
00:09:04.21 Our dream approach is. the basic feature of oncogenic Ras is that is cannot hydrolyze GTP.
00:09:12.09 Can we think about making small molecules that would induce GTP hydrolysis on oncogenic Ras?
00:09:18.21 This is probably a dream project and we're still working on it to make it feasible.
00:09:25.05 But I will show you, in the course of my seminar presentation, that the reason we still think its possible
00:09:32.23 is that the chemistry of it should not be so difficult.
00:09:36.14 And I hope I can convince you and that you will go with me on that point in the end.
00:09:40.26 So, we are talking about, here, a nucleophilic attack of water on the gamma phosphate of GTP
00:09:49.03 mediated by RasGAP and it produces Ras-GDP and Pi. A simple biochemical reaction
00:09:56.09 but if it doesn't work it leads to very drastic consequences, namely cancer.
00:10:00.21 So first of all (And you have seen that picture also a number of times.
00:10:05.24 This is my introductory slide, always)
00:10:08.09 is that the two amino acids that are most frequently mutated
00:10:13.16 (either of them) in oncogenic versions of Ras, they are very close to the active site.
00:10:18.07 So you see the nucleotide here: that is the gamma phosphate
00:10:22.07 And you see this is glutamine 61 and this is glycine 12 are very close to the active site
00:10:28.10 obviously, as you would probably predict or thought of before,
00:10:32.06 is that they are somehow involved in the GTPase reaction.
00:10:35.22 Now, we'll obviously show you what they actually do and why the mutation leads to a block in GTP hydrolysis.
00:10:43.08 And you also can see here from a surface representation of the Ras active site
00:10:49.12 that if this GTP. so it sits. its bound to the surface
00:10:53.26 and you see the gamma phosphate is still approachable from the outside
00:10:57.03 and that will be important in the context of what I will be talking about
00:11:01.12 and you see also, I talked, last time about magnesium being important.
00:11:05.20 If there is no magnesium around you also have no GTP hydrolysis reaction.
00:11:09.24 So, as always in a mammalian genome, there are not just one RasGAP, but rather, 12 or 13.
00:11:19.08 And I indicated here a few of these.
00:11:24.06 And you see they all contain one particular domain of about 330 residues.
00:11:28.09 This is the RasGAP domain which by itself is able to initiate the fast GTP hydrolysis reaction.
00:11:36.17 And you see that all these proteins are composed of different domains.
00:11:43.03 And some are similar, some are vastly different.
00:11:45.20 And they obviously are regulating Ras in different contexts of the cell
00:11:50.04 due to their different domains that they have in addition.
00:11:53.15 And I will be talking about one, the first RasGAP to be discovered by Frank McCormick which is P120GAP.
00:12:02.06 And I will be talking later on about NF1
00:12:05.05 which is another important element for tumor formation because it is a tumor suppressor gene.
00:12:12.04 So first of all, and again to remind you, the intrinsic GTP hydrolysis is very slow.
00:12:17.23 But, if you add the GTPase activating protein, it is fast.
00:12:21.08 So what I have been doing here is I take radioactive GTP (gamma labelled, for example)
00:12:25.27 and then I measure the production of Pi over time.
00:12:29.26 You see, down there, this reaction (this is at room temperature) is almost negligible.
00:12:35.14 There's almost no hydrolysis of normal Ras at room temperature without GAP.
00:12:40.12 And if you now add a particular amount of RasGAP you see that there is a very fast reaction.
00:12:44.23 Much much faster.
00:12:46.04 And under limiting conditions it's actually about 10^5-fold stimulation of that reaction.
00:12:53.10 So that is a way we want to look at it.
00:12:57.05 We want to analyze how GAP mediates this fast GTP hydrolysis reaction.
00:13:03.14 So, initially, obviously, you ask yourself: What could be the mechanism of hydrolysis
00:13:10.11 and what is the step that is catalyzed by GAP?
00:13:14.02 You can think of Ras-GTP being, in principle, a fast GTP hydrolysis enzyme
00:13:20.14 but it needs to come into a GTPase competent state by an isomerization reaction.
00:13:26.15 And that is very slow and is catalyzed by GAP.
00:13:28.29 Or you could think that the actual cleavage reaction, going from GTP to GDP Pi,
00:13:35.02 is very slow and is catalyzed by GAP.
00:13:38.01 Or you could even think that all of that is still very fast
00:13:41.09 but the release of Pi to product is very slow and that is catalyzed by GAP.
00:13:47.08 For example, if you remember having heard about the hydrolysis of ATP on myosin,
00:13:53.26 myosin is, for example, a very fast GTPase but the Pi release is very slow
00:13:59.00 and needs to be catalyzed by actin.
00:14:01.04 So, in other words, what is the actual step that is catalyzed by GAP in this particular case?
00:14:07.10 And I should say that it is the cleavage reaction itself
00:14:11.12 which is the major point of attack by GTPase activating protein
00:14:16.21 and that only in the presence of GAP do the other reactions
00:14:20.18 become at least partially rate limiting, at least the Pi release.
00:14:24.10 And I'll show you that later on, as well.
00:14:27.29 And although I've shown you that before, I will repeat that again.
00:14:32.12 So what we use a lot in our studies where we do biochemistry with fluorescent nucleotide,
00:14:38.20 we use a mant- or mGDP or mGTP analog
00:14:42.29 where you have, on the ribose, a fluorescent reporter group.
00:14:46.24 So this would be either deoxy ribose or ribose and on the ribose you have bound by
00:14:52.17 these ester bonds, the mant group which is very sensitive to the environment that it sits in.
00:14:59.19 And that's why it always give very beautiful structural changes as I will show you in a minute.
00:15:07.10 We used stop flow for measuring fast reactions because, remember, the GTPase reaction,
00:15:13.29 which is slow in the absence of GAP becomes very fast
00:15:17.15 and so in order to analyze it in detail, we need to use stop-flow kinetics.
00:15:21.16 So what you'll, for example, do: you have Ras labeled with
00:15:25.10 mant-GTP (so it's the fluorescent version of GTP)
00:15:28.16 and you have GAP and you shot them together into a fluorescent-detection cuvette
00:15:34.02 and you have the stop flow up here in order to
00:15:37.17 make sure that you only put a certain amount of liquid from these two syringes into your reaction chamber.
00:15:44.07 So when we do that, when we shot these two things together, we see that there is
00:15:50.09 a fluorescent increase if you use Ras-mantGTP and a decrease.
00:15:54.09 The increase, again, is very fast.
00:15:56.25 It means the two proteins make a complex.
00:15:58.27 And then over time, the complex dissociates because after hydrolysis neurofibromine does not bind to Ras anymore.
00:16:07.18 And all of this is over, as you can see, after one second.
00:16:10.09 So, very fast phospho-transfer reaction in the presence of saturating amounts of GAP.
00:16:15.07 And as a control, we use Ras bound to an analog, GppNHp,
00:16:21.25 where you that between the beta and the gamma phosphate you have an NH group
00:16:25.16 which cannot be hydrolyzed anymore and now you have, also, a very fast increase
00:16:30.17 (which is complex formation) but no dissociation because that cannot be hydrolyzed.
00:16:35.05 So that is also the proof that the dissociation is due to GTP hydrolysis.
00:16:40.21 So one may ask, "In this reaction here, what on GAP, which residues on GAP,
00:16:50.01 are involved in mediating this fast GTP hydrolysis reaction, that 10^5 fold stimulation of the reaction?"
00:16:58.17 And obviously, you were thinking of which residues, which amino acids could do the job.
00:17:03.16 Or is it more than one amino acid?
00:17:05.16 And as a good candidate, we were thinking of an arginine
00:17:08.16 because if you look at another G-protein, G-alpha protein (of the heterotrimeric G proteins)
00:17:15.02 it is known that that consists of two domains
00:17:18.25 so this blue domain is the G-domain and the red stuff here is a helical extra domain.
00:17:24.21 But in that helical domain you have an arginine which sits right, smack in the active site
00:17:29.24 and it has been shown that that arginine is important for the reaction.
00:17:33.29 And that has been shown, in a very old experiment
00:17:37.20 because there is a bacterial pathogen called Vibrio cholera which induces cholera
00:17:45.29 and what is does is it actually modifies this arginine on this G-alpha protein.
00:17:51.16 You see, you have NAD and the cholera toxin transfers ADP-ribose onto
00:17:58.03 an arginine of G-alpha which is shown here. This is actually from the textbook here.
00:18:02.26 So this a very old reaction. It had been analyzed many, many years ago.
00:18:07.10 And it shows, when you do this reaction, when you block
00:18:10.16 the GTPase reaction on G-alpha protein, there is no GTP hydrolysis anymore
00:18:16.10 and the protein is now producing cyclic AMP,
00:18:19.14 it opens up channels (cyclicAMP-gated ion channels) and then you get all these symptoms of cholera.
00:18:26.19 So again the question is: would arginine be a good candidate?
00:18:29.29 So we looked for conserved arginine in all the RasGAPs that I've shown you
00:18:33.24 and indeed, we found several, most of them didn't make any difference.
00:18:38.06 But there was only one arginine that when mutated shows the following pattern in the reaction.
00:18:43.19 So you, again, have in the green version (this is the wild-type version)
00:18:47.23 fast increase due to complex formation, fast decrease due to dissociation after GTP hydrolysis.
00:18:53.16 And with these mutants here, arginine mutated to either lysine or alanine
00:18:58.11 or anything you'd like to mutate it to,
00:19:00.03 there is again an increase in the reaction but then it stays up there, no hydrolysis whatsoever.
00:19:06.13 Or, at least under those circumstances, up to a second, there is no hydrolysis.
00:19:10.16 Which tells you already, obviously, that this arginine must be essential for the reaction.
00:19:18.02 So far so good. The biochemistry was clear
00:19:21.09 but obviously you don't really know what arginine is doing in the context.
00:19:24.05 You think you have an idea it may be going into the active site
00:19:28.02 but, again, we have to wait for the structure to tell us really what it does.
00:19:33.21 First of all, though, let me introduce you to another important concept for analyzing phospho-transfer reaction.
00:19:40.12 And that is using aluminum fluoride complexes.
00:19:43.02 You may wonder why such an inorganic molecule would be important
00:19:47.14 but it turns out that if you look at the nature of the transition state,
00:19:51.20 so this would be GTP being attacked by water.
00:19:55.18 You have a transition state where the phosphate now makes a triginal, flat thing
00:20:00.26 where the nucleophillic oxygen and the leaving group oxygen on the other side
00:20:08.22 are the axial ligands of this penta-coordinate phosphate
00:20:12.11 and this transition state can either be very tight (which is then called associative)
00:20:18.16 or very loose depending on the distance here between phosphate and the two axial ligands.
00:20:25.26 And then you end up with, again, tetragonal phosphate (this Pi free phosphate).
00:20:32.08 And it turns out that aluminum fluoride
00:20:36.05 (which has distance between aluminum and fluoride very similar to
00:20:39.23 between phosphate and oxygen and they are also highly electron negative)
00:20:44.26 that this is a very good mimic of the transition state of the phospho-transfer reaction.
00:20:51.02 The only problem was that while, for example kinesin or myosin or
00:20:56.28 many other phospho-transfer enzymes use aluminum fluoride as a mimic of the
00:21:02.07 gamma-phosphate in the transition state,
00:21:04.17 Ras or Rho and all of these Ras-like proteins, never showed that.
00:21:08.25 Here you see that experiment.
00:21:11.19 You take Ras-mantGDP (it has a certain fluorescent emission spectrum with a maximum at around 440)
00:21:19.03 and now you add the GAP and with one there's no change whatsoever to the spectrum.
00:21:24.12 So nothing happens.
00:21:25.11 But if you now add aluminum fluoride, you see that now you get a blue shift first of all
00:21:31.00 and then an increase in the absorption.
00:21:37.11 That means there is a trimeric complex between Ras, NF1 and aluminum fluoride
00:21:42.07 where the aluminum fluoride sits in the gamma phosphate binding site.
00:21:47.10 And that was very instrumental.
00:21:48.25 and by the way, if you now do this in oncogenic mutants
00:21:52.02 which we know does not hydrolyze GTP and do the same experiment,
00:21:55.15 you see, in the presence of aluminum fluoride and Ras, there's no fluorescent change.
00:22:00.06 And now you add NF1 and there's no increase in fluorescence
00:22:04.12 because you have a mutation (Q61L) that does not hydrolyze.
00:22:08.13 You can also take a mutation of NF1, for example with the arginine mutation,
00:22:13.22 and again there would be no change whatsoever.
00:22:16.05 So in other words, what these experiments again tell us is that Ras in an incomplete phospho-transfer enzyme.
00:22:22.22 It needs the presence of a GAP in order to look like a phospho-transfer enzyme
00:22:28.05 and if there's any mutation that blocks the GTPase reaction, either on Ras or on NF1,
00:22:33.22 we also get no aluminum fluoride complex.
00:22:37.09 So obviously, all of this is nice in terms of doing biochemistry
00:22:42.00 but what really mediates the GTPase reaction, the fast one,
00:22:47.22 we think can only be verified by looking at the structure of the Ras and RasGAP complex.
00:22:55.29 This is shown here. So that was the paradigm for analyzing this type of reaction.
00:23:02.06 So you see, for example, in red you see Ras and down in green is the RasGAP domain (P120 GAP).
00:23:09.13 And what you see then, here. if you look very carefully
00:23:12.04 you see that there is a residue from GAP going into the active site.
00:23:17.14 You see that when it comes back. you see that right there.
00:23:21.01 There is an arginine residue pointing into the active site of Ras
00:23:26.04 and what is does is shown in the next slide.
00:23:29.06 First of all, in this slide it shows the aluminum fluoride really is the flat triangle
00:23:34.00 that sits between the leaving group and the nucleophillic water.
00:23:38.07 So there is a mimic of the transition state.
00:23:39.28 And you see then, also, what are the residues that mediate fast GTP hydrolysis.
00:23:45.17 So this would be the phosphate,
00:23:48.14 so we think now that if we take away aluminum fluoride and
00:23:52.06 think of what the real transition state would look like
00:23:55.06 you have the nucleophillic water and you have the transition state
00:23:58.29 where they are bound to the gamma phosphate
00:24:00.25 and the glutamine is fixating the water relative to the phosphate
00:24:05.07 by an acceptor and a hydrogen bond donor interaction.
00:24:09.17 And the arginine (what we call arginine finger) is first of all stabilizing
00:24:13.29 the position of that glutamine and it also delivers this positive charge
00:24:18.03 of the amino group to neutralize charges in the gamma phosphate.
00:24:22.28 So these are the two residues that are really crucial for the reaction
00:24:27.08 glutamine 61 from Ras and the arginine finger from RasGAP.
00:24:32.05 And this already explains, for example, why mutations of glutamine 61 in oncogenic Ras
00:24:37.19 mess up the hydrolysis reaction because you can imagine if you have, for example,
00:24:42.09 a leucine or whatever here, if cannot do this kind of interactions here.
00:24:47.00 So any mutation of glutamine 61 is oncogenic because it is a direct catalytic residue.
00:24:53.16 So the structure also explains why mutations of glycine 12 are oncogenic.
00:24:58.22 And that also can be easily seen here on this slide.
00:25:02.04 You have glycine 12 which when mutated to any residue makes it an oncogene.
00:25:06.25 And then you see aluminum fluoride being this flat triangle.
00:25:11.08 You see glutamine 61 being there and you see the arginine finger down here.
00:25:16.09 And they're all very very close together indicated by these blue dashed lines
00:25:20.04 which are indicating that these are almost VanDerWaals distance.
00:25:24.04 And now if you mutate, for example, glycine to the smallest possible amino acid,
00:25:28.11 it would be alanine, you see that it would immediately mess up all these residues in the active site.
00:25:34.01 So for steric reasons, there can be no other residue
00:25:36.24 in the active site in the transition state except for glycine.
00:25:43.08 So the message from all of this was, obviously, that there's an arginine
00:25:49.00 that we call the arginine finger that pulls the trigger on the GTPase reaction.
00:25:54.00 Without it, it is very slow and with it, it becomes 10^5 fold faster.
00:26:01.01 We used also. so coming back to the question: what is now rate limiting in the whole process?
00:26:07.00 So the main regulating step of intrinsic GTP hydrolysis is the very slow chemical step, the slow hydrolysis.
00:26:14.29 But using a different technique we now find out that
00:26:17.25 there is another step that become partially rate limiting and I will show you that in a minute.
00:26:23.29 So we use time-resolved Fourier transform infrared spectroscopy.
00:26:28.10 And with that we can do almost atomic resolution.
00:26:33.11 We can look at atoms in the active site on a millisecond timescale.
00:26:37.27 So if you see an infrared spectrum of a protein, which has amide I and amide II bands,
00:26:47.20 which is not very structured information because its a mixture of information on
00:26:54.26 alpha helices, beta sheets and so on and not a great deal of detail can be taken from such a picture.
00:27:02.26 But what we do is we observe, for a reaction. let's say a protein goes from A to B
00:27:09.05 we observe the different spectrum which at least on this absorption scale
00:27:16.19 does not show much of a difference but if you look in more detail.
00:27:20.15 so you see the absorption there is 0.0 and the absorption down here is 0.02,
00:27:25.14 so we see very small but very reproducible changes
00:27:29.21 in the course of the reaction when A goes to B.
00:27:33.02 And we see negative peaks that mean A goes away and we see positive peaks if B comes up.
00:27:40.22 So we can observe things that are lost and things that come up
00:27:43.29 in the course of the reaction and we can follow these by FTIR.
00:27:49.25 And so if you do. so first of all we need to trigger the reaction at a particular time point
00:27:56.25 in order to observe second timescale structural changes.
00:28:03.04 And in order to do that we use, again, a different analog
00:28:07.00 and the different analog is caged GTP which was developed by Roger Goody
00:28:10.22 who is a colleague of mine in the Institute.
00:28:14.05 And this caged GTP is blocked on the gamma phosphate by what is called the cage group.
00:28:20.22 So it doesn't allow hydrolysis but now, with a flash of light, you can cleave that cage group off
00:28:26.21 and now you have Ras-GTP which can then hydrolyze to GDP and Pi.
00:28:31.19 If you do that with let's say Ras without GAP,
00:28:39.11 what you see is that you have a. for example, you see absorbance.
00:28:44.22 so any absorbance that you might know from infrared, shows an atomic vibration in the bonds.
00:28:51.09 For example, you see vibrations for the
00:28:53.15 alpha, the beta, the gamma, which decrease toward the end and become zero after 2 hours, 5 minutes.
00:29:00.29 So the difference spectrum (subtract the end spectrum from the starting spectrum)
00:29:06.16 and then you see only the changes that are occurring during the reaction.
00:29:11.23 And that is quite normal. There is a single exponential decay when Ras hydrolyzes GTP.
00:29:18.21 No big deal.
00:29:20.15 But what is interesting is that when you analyze the GAP mediated reaction
00:29:24.26 because now you don't see a single exponential decay, but you suddenly see intermediates appear.
00:29:30.14 You see peaks that come up and go down and the most important one is
00:29:35.04 indicated by this number 1113. This is the frequency for that particular change.
00:29:39.28 So you obviously have to analyze what is each band doing, what does it belongs to.
00:29:47.06 So what we're doing in order. we have developed these techniques
00:29:52.17 or our colleagues at the University of Bochum with whom we collaborate
00:29:56.03 they have developed techniques to find out what is each bandwidth,
00:30:01.01 what is each frequency. absorbance change. what is it due to.
00:30:03.28 For example, you find for GTP that there is absorbance change coming up with
00:30:10.13 a rate constant k2. So k1 is the photoisomerization, k2 is one reaction and k3 is the next one.
00:30:17.18 And if you do that, you get an increase and a decrease with k3.
00:30:21.12 And then there is an intermediate which is coming up at 1113.
00:30:26.12 It comes up with k2 and decays with k3.
00:30:30.01 There is free Pi coming with k3. From all of that we can obviously conclude
00:30:35.18 that there is a Pi intermediate with this absorption frequency here, 1113 cm^-1
00:30:44.02 which appears with the rate constant k2 and decays with a rate constant k3.
00:30:50.03 So in other words, the release of Pi is now becoming visible.
00:30:54.25 So we see hydrolysis when this Pi peak comes up.
00:30:58.21 And we see decay when it goes away.
00:31:01.01 And you see this here, for example, in real-time.
00:31:03.25 So the absorption change with the rate constant k2. you see the scheme here:
00:31:09.05 Ras when its in the on state goes to the Pi state and you see there is a protein bound Pi
00:31:17.13 that comes up and the Pi band goes down in the course of the reaction.
00:31:21.20 And this is repeated a number of times.
00:31:23.07 And you also so that there is an arginine finger that is coming in and out of the reaction chamber.
00:31:30.00 So we can follow not just protein bands,
00:31:32.16 we can follow the phosphate bands at atomic resolution on a milli second timescale.
00:31:40.26 And that tells us now, all together, that is the message from all of this,
00:31:46.01 that while you have a high activation energy for the intrinsic hydrolysis reaction of 92kJ/mol
00:31:54.01 you now seperate the reaction in two partial reactions,
00:31:57.16 which have a lower activation energy and that's why is makes it so much faster.
00:32:01.08 So you have the first activation energy for the cleavage reaction. which leads to Ras-GDP-Pi.
00:32:08.03 And you have the second step where you have release of Pi and now you have the product.
00:32:12.14 So that is, again, a general theme of enzymology
00:32:15.12 that an enzyme catalyzed reaction lowers the activation energy
00:32:20.00 not just by lowering one reaction but also by subdividing it into partial steps
00:32:24.28 each of which has a different activation energy.
00:32:29.22 And here you see that this activation energy is 59 kcal/mol and 66 which means
00:32:36.04 that this is a bit higher and that's why this is the partially rate limiting step of the overall reaction.
00:32:45.12 So let me now give you. so that was Ras and how it leads to tumor formation
00:32:50.14 and we analyzed in detail how this function even on a biophysical level
00:32:55.02 but now let me come back to why certain GTPase reactions, when the don't work,
00:33:03.00 how they lead to different types of diseases.
00:33:05.20 Neurofibromatosis is one of them.
00:33:07.27 I already showed you that neurofibromine, the gene product of the gene is a Ras GAP.
00:33:15.09 And there is a disease called Type I Neurofibromatosis. Its what people have cafe au lait spots on the skin,
00:33:22.21 sometimes small tumors on the skin which can sometimes be rather large and very disfiguring
00:33:28.16 which are caused by mutation of deletion of neurofibromatosis gene.
00:33:35.21 And for example, when we worked on the mechanism of GTP hydrolysis, by GAP and NF1,
00:33:43.00 a colleague from the Charite Clinic in Berlin came to us and told us that he had
00:33:48.03 a female patient which dies at teh age of 35 and she has three sons indicated up there
00:33:55.20 which also have the disease. And he analyzed the blood of the patient and then the tumor itself.
00:34:04.22 He found out that there is a mutation in the sequence of neurofibrobromine
00:34:11.29 where the arginine is mutated to proline. You see that down there arginine is mutated to proline.
00:34:17.13 And obviously I wouldn't tell you that, if it wasn't the catalytic arginine.
00:34:21.19 So it turns out they have a mutation in the catalytic arginine
00:34:25.23 and when you now do the GTPase reaction that I've shown you before,
00:34:30.09 you see that you have, with normal NF1 you have the blue curve here,
00:34:37.17 it means an increase in fluorescence and a decrease with hydrolysis
00:34:43.03 and now if you take this mutation, R to P, you have increase which means complex formation
00:34:48.03 but no hydrolysis and there is another patient that we've analyzed in the meantime
00:34:53.16 again arginine to anything else, Q in this particular case,
00:34:58.09 leads to a block of its ability to hydrolyze GTP on Ras.
00:35:02.17 So mutation of the essential arginine leads to the disease neurofibromatosis.
00:35:08.16 Also, I should point out that there are many other mutations in neurofibromine
00:35:13.22 which also cause the disease which phenotypically very different in many different patients.
00:35:20.26 Let me introduce you now to a different system that is
00:35:24.06 interesting both from a biochemical standpoint but also from a standpoint of a different disease
00:35:29.11 that I will come to in due course.
00:35:33.03 So what I will be talking about is a molecule called Rap which, obviously, has a cognate RasGAP.
00:35:40.16 And Rap is close homolog of Ras and the name derives from
00:35:44.05 the fact that it is highly homologous to Ras because Rap stands for Ras Proximate.
00:35:49.07 Even though it was considered to be a close homolog of Ras, it does something completely different.
00:35:55.14 It obviously has the same cycle between GDP and GTP. So it works as a molecular switch.
00:36:02.00 But it does not have to do with proliferation, as Ras, or differentiation, but rather
00:36:06.10 it is involved in integrin activation, or platelet activation or other things.
00:36:11.19 So the biology of Rap is completely different.
00:36:14.17 Why the biochemistry is so interesting is that Rap is the only homolog
00:36:22.12 or the only member of the Ras super family that doesn't have a glutamine in the position
00:36:27.18 where glutamine 61 of Ras is involved in GTP hydrolysis.
00:36:33.01 So, it misses the residue that we thought was absolutely crucial GTP hydrolysis and here, its not there.
00:36:39.18 And, obviously, the question is why that is so and how does RapGAP
00:36:42.25 then work on this system and how does it stimulate the reaction.
00:36:48.29 So first of all, let me introduce you to the RapGAPs.
00:36:52.24 There are indicated here five of them but there are more in the human genome
00:36:57.23 but these five RapGAPs all contain a domain that is highly homologous
00:37:04.02 which is indicated here by the light blue and the dark blue staining
00:37:08.09 where the light blue stuff is somewhat different to the dark blue.
00:37:12.05 And I will explain that when we look at the structure.
00:37:13.21 So again, the domain organization of all these five RapGAPs is somewhat different.
00:37:17.25 That means they're probably doing their job in a different biological context.
00:37:23.04 The reason its also interesting is that the dark blue homology region is
00:37:28.10 also conserved in a protein called Tuberin
00:37:30.17 which stands for a disease that I will be talking about in the end.
00:37:34.03 So that's why it was also interesting to look at that reaction.
00:37:37.06 And the third reason its interesting to look at that reaction is indicated in the next slide.
00:37:42.19 But before doing that, let me show you first of all that we do, again, a stopped-flow fluorescence assay.
00:37:48.23 We've developed a system where we can look at this reaction in a biochemical way
00:37:53.12 and analyze mutations and the speed of reaction and so on.
00:37:58.13 So here again, we have Rap-GTP interacting with RapGAP
00:38:03.20 You get a large, quick fluorescent increase which is due to complex formation
00:38:07.28 and a decrease due to GTP hydrolysis and dissociation of the product.
00:38:12.25 And it is all over, again, after one second.
00:38:16.03 whereas without RapGAP, the whole reaction would take hours.
00:38:19.25 So, in other words, we again have 10^5 stimulation of the reaction.
00:38:23.15 But the reason this is also an interesting system biochemically and mechanistically,
00:38:29.09 is that there are a number of conserved arginines in RapGAPs and obviously
00:38:35.00 we thought that one of them would be involved in providing an arginine finger to the system.
00:38:39.06 In fact, we mutated all of them to alanine
00:38:42.06 and none of them has any dramatic effect on activity as one can see here.
00:38:48.02 So the worst reaction is still close to .5/second.
00:38:52.02 So in other words, there is not a dramatic effect when you mutate an of the arginines
00:38:55.29 which makes it unlikely that there is an arginine finger involved in the reaction.
00:39:00.23 So the two residues, intrinsic glutamine and the arginine finger in trans
00:39:09.04 which is Ras and Rho and others make the important catalysis are not here in this system.
00:39:17.05 That means the whole chemistry must be totally different.
00:39:21.15 So we looked at the FTIR of the system again and
00:39:26.07 it shows basically the same features even thought their structures are somewhat different.
00:39:31.16 The basic features are that there is a Pi intermediate whose decrease is rate limiting
00:39:37.23 for the reaction and although its chemically somewhat different,
00:39:41.27 as the different absorption spectra show, it is, indeed, kinetically a most important intermediate.
00:39:50.11 But what was interesting, and we found in this particular case, in the RapGAP case,
00:39:56.10 and it was also found in other systems in the meantime,
00:39:58.27 is that the GTPase reaction is reversible,
00:40:01.29 which sounds sort of crazy because it's a downhill reaction.
00:40:05.06 If you take GDP and Pi and the whole system you would never ever create GTP.
00:40:09.22 But what happens is that if you analyze the reaction by
00:40:13.05 using, instead of normal water, O18 water, which is indicated by this black dot,
00:40:17.23 you would expect that in the reaction you get hydrolysis and then you get GDP and Pi
00:40:23.27 which has one phosphate labelled as O18 oxygen-rather, one oxygen labeled as O18 oxygen.
00:40:30.12 But instead of getting a Pi with one O18 oxygen, you get Pi with two O18 oxygens,
00:40:36.24 with three O18 oxygens, and with four O18 oxygens as analyzed here by a mass spec.
00:40:42.11 You see where you get all these four products, analyzed by mass spectroscopy.
00:40:49.27 So, how does this happen?
00:40:51.09 So it happens because on the protein you have this long-lived intermediate
00:40:56.00 with GDP and Pi, sitting in the active site before going into product that can also
00:41:03.10 do a back reaction to make GTP with one oxygen now on the gamma phosphate.
00:41:08.15 Now, if it reacts again with O18 water, you get incorporation of a second O18 into the product.
00:41:15.27 And if it happens again, a third one and a fourth one.
00:41:18.16 So although the overall reaction is downhill,
00:41:22.12 on the enzyme, you're getting a backwards reaction.
00:41:24.15 And you can never get it once Pi is released
00:41:28.21 because then the activation energy for the reverse reaction is too high.
00:41:33.18 So we solved the structure of RapGAP also.
00:41:37.06 This is a two domain structure where one domain, which you see now to the left,
00:41:44.07 is the catalytic domain--the dark blue region in the homology diagram that I showed you
00:41:49.01 and the light blue stuff is the dimerization domain which is not important for catalysis
00:41:53.08 but is there to dimerize the protein
00:41:56.18 for whatever reason that we really do not know.
00:42:00.13 So in analyzing the catalytic domain, obviously, you ask yourself:
00:42:03.10 What are the conserved residues and which of those play an important role in catalysis?
00:42:08.04 The purple helix that I've indicated here is the most highly conserved region.
00:42:12.00 So it's likely that residues on this helix are somehow involved in catalysis.
00:42:18.05 And indeed, you can mutate many of these and you see certain effects.
00:42:21.12 But the most dramatic effect happens when you mutate
00:42:25.01 N290, so an asparagine, sitting on this catalytic helix.
00:42:29.08 When you mutate that, you get the following result.
00:42:33.00 So we call it, by the way, the Asn-Thumb to make a difference to the Arginine Finger.
00:42:39.03 So it's an Asn-Thumb and you'll see in a minute why we call it an Asn-Thumb.
00:42:44.26 So, first of all, if you now look at the mutation that I talked about,
00:42:48.27 so if you take the N290A mutation and analyze the reaction by, again,
00:42:55.09 this fluorescent stopped-flow assay, you see that while wild type makes a complex
00:43:00.25 and then decays into product,
00:43:02.26 the mutation here makes a complex, this is even, by the way, tighter than in the wild type case,
00:43:09.15 but there's absolutely no hydrolysis.
00:43:11.07 The reaction goes on and on. It stays up there and never goes down.
00:43:14.21 Whereas if you make a mutation like H287 to alanine,
00:43:20.22 you see that there is no complex formation because the fluorescence stays down here.
00:43:24.04 So that reaction is dead because it cannot bind
00:43:27.08 but the red reaction is deal because we think that this is the important catalytic residue.
00:43:32.12 We think that from looking just at the biochemistry.
00:43:35.15 Obviously, to know what it does, we need again to solve the structure,
00:43:38.26 which we did. This is the Rap-RapGAP complex.
00:43:43.15 And you see here the red and the green stuff is RapGAP and the blue stuff is Rap.
00:43:49.20 And you see GTP. But what you also see if you look at it in detail,
00:43:54.02 is that there is, again, something pointing from the red catalytic domain of RapGAP
00:43:59.06 into the active site and that is an asparagine.
00:44:01.17 Obviously, it's the asparagine that I've talked about.
00:44:04.20 which pokes into the active site of Rap. That's why we call it the Asn-Thumb
00:44:08.28 in relation to the Arginine Finger in the other systems.
00:44:12.27 And if you look in detail at what happens at the active site,
00:44:17.02 and if you compare it to three other structures of Ras protein and their cognate GAPs,
00:44:26.19 taking Ran and RanGAP, Ras and RasGAP, and Rho and RhoGAP,
00:44:31.18 where Rho and RhoGAP are from Rittinger et al. and the other structures are from us,
00:44:35.14 you see that the other three, these three structures here,
00:44:40.12 have a glutamine pointing towards the catalytic water,
00:44:44.29 which attacks the gamma phosphate indicated here,
00:44:47.09 which is the aluminum fluoride,
00:44:50.01 a transition state which mimics the gamma phosphate that is transferred.
00:44:54.08 And the red structure here has, instead of the glutamine 61, has a threonine
00:45:00.19 which is pointing aways from the active site has nothing to do with catalysis.
00:45:04.01 Instead, what you see is that the purple helix here inserts this asparagine
00:45:09.19 into the active site just exactly at the position where the others have the glutamine.
00:45:14.23 So the differences here: the three other structures have a glutamine, which is in cis
00:45:19.29 and Rap and RapGAP have an asparagine in trans
00:45:23.17 which does the same thing namely, stabilizing the catalytic water.
00:45:30.04 And if you look at the surface of the protein,
00:45:34.01 this is Rap surface shown as a surface representation,
00:45:39.07 and added to it is just the helix from RapGAP.
00:45:43.09 It sits on the surface and inserts this asparagine into the active site.
00:45:48.13 So the gamma phosphate peaks out of that hole.
00:45:50.08 And all RapGAP really does is it has on the helix this Asn, it puts it into the active site,
00:45:57.17 and that alone seems to be, at least in a chemical sense,
00:46:03.03 responsible for stimulating the GTPase reaction by 10^5 fold
00:46:07.10 because if you mutate that thing, it still binds alright, but there's absolutely no hydrolysis.
00:46:11.22 So that makes us think about, again, the future of designing anti-Ras drugs.
00:46:18.02 If all it takes is to insert such a residue into the active site,
00:46:22.11 from a chemistry point of view it should be doable,
00:46:24.24 but we obviously have to develop molecules that bind in a correct way
00:46:28.12 onto the surface, which is not so easy and we are still thinking that we might be able to do it.
00:46:35.15 This is, again, coming back to that.
00:46:38.00 So as an approach to anti-cancer drug target,
00:46:42.03 let's find molecules that induce hydrolysis of oncogenic Ras.
00:46:46.20 And from what we have observed with RapGAP we think, we have hope that it can be done.
00:46:54.26 And the third reason why working with the Rap-RapGAP system is that
00:47:03.05 it's connected to a disease called Tuberous Sclerosis, a benign tumor.
00:47:06.19 People come in with hamartomas in many organs.
00:47:09.16 But the most obvious feature and what the name comes from is that
00:47:14.07 people have, when they do an NMR of the brain, they have
00:47:16.29 these sclerotic, tuberous sort of things in the brain that you see in the NMR of the brain.
00:47:24.17 The people have mutation in two proteins called Tuberin and Hamartin.
00:47:30.22 And Tuberin, as I showed you before, has high homology
00:47:34.22 to RapGAP in this dark blue region, this means the catalytic region.
00:47:39.07 And if you now look, for example, where patient mutations in Tuberous Sclerosis,
00:47:44.23 in Tuberin, where they are occurring.
00:47:47.00 And this is an alignment of different RapGAP sequences and they align with Tuberin sequence.
00:47:53.27 You see that most highly conserved region is, again,
00:47:57.09 this thing here (the red stuff) where you have the catalytic helix.
00:48:01.08 And one of the mutations in a patient with Tuberous Sclerosis
00:48:04.22 (and many actually have that mutation) is on
00:48:07.06 this asparagine that we know is the important catalytic residue.
00:48:11.08 So this is another version of what we have seen before
00:48:14.21 an important catalytic residue is mutated in the disease Tuberous Sclerosis.
00:48:21.19 So let me, in the last five minutes, just give you another disease,
00:48:25.22 just to make sure that it's not just the Ras and Rap system,
00:48:28.28 that there are many diseases where the inability to hydrolyze GTP leads to very many different diseases.
00:48:35.07 So this is Retinitis pigmentosa.
00:48:38.28 People have pigments on the retina. That's what the name comes from.
00:48:42.28 And they lose vision, they lose peripheral vision.
00:48:47.00 So this is a normal person seeing that building and this is a patient with the disease
00:48:52.15 that loses more and more, progressively, his peripheral vision.
00:48:56.00 And then loses complete vision after the disease develops fully.
00:49:01.19 And there are a number of genes mutated in Retinitis pigmentosa
00:49:06.12 that are called RP12x and whatever.
00:49:09.28 And there is one form that is called RP2.
00:49:13.03 It's an X-linked disease where people have mutations in. a lot of point mutations in a protein.
00:49:21.16 We determined the structure of the protein, which looks like this.
00:49:24.24 This is a beta helix domain, and this is another domain.
00:49:27.20 And you see many of the mutations that you then analyze.
00:49:31.06 You find out that what you see is that most of the mutations
00:49:37.16 determine or mess up, probably, the structure because they are inside
00:49:41.24 the hydrophobic core of the protein and you would imagine that
00:49:45.04 they don't do anything to the catalysis or the interaction of this protein.
00:49:50.19 But there are a few mutations, indicated here, for example,
00:49:54.01 E138G or R118 to H, K, or C. So these residues point into solution.
00:50:03.27 So you would think that they do something important for interaction of the protein
00:50:08.20 or in something--that they are involved in what these proteins are normally doing.
00:50:14.20 So we solved, again, the structure of the complex between Arl3.
00:50:19.13 So we knew that RP2 interacts with Arl3, another Ras-like protein.
00:50:24.26 It's called Arl because it's an Arf Related Protein.
00:50:28.04 And we solved the structure of that. So you see this stuff here would be the RP2 in purple.
00:50:37.25 And you see in green would be Arl3 in a GTP bound form.
00:50:41.25 And what you see then, if you, again, look very closely,
00:50:44.26 you see that there is a residue from RP2, right here, which points into the active site of the Arl.
00:50:52.10 So one didn't know what RP2 was doing, but when we solved the structure,
00:50:55.17 we could immediately see that it smells like a GAP because
00:50:59.18 it puts an Arginine Finger into the active site of the other protein.
00:51:05.08 And if you look at the active site in detail you see that this would be GDP, again,
00:51:10.28 this is aluminum fluoride, this is glutamine from Arl3,
00:51:15.14 and these are three residues from RP2--Q116, R118, E138.
00:51:21.26 And, obviously, all of these residues are mutated in retinitis pigmentosa.
00:51:27.15 And you can see that the arginine is doing the same thing that you've seen now many times before.
00:51:32.22 And it is stabilized by these other residues and all three residues, when mutated,
00:51:38.10 mess up the GAP activity of RP2.
00:51:41.26 So here, the structure really told us what the protein is doing.
00:51:44.19 There was no idea about the function and the structure told us, exactly, that this is a GAP for Arl3.
00:51:53.17 So let me now come to the conclusions from what I have been telling you about.
00:52:00.12 First of all, maybe, conclusions about Ras itself because it's the most important oncogene.
00:52:06.14 It's an incomplete enzyme, it cannot hydrolyze GTP very fast.
00:52:11.09 But then comes RasGAP which stabilizes switch II and the glutamine 61,
00:52:16.10 which is the important structural catalytic element and it also supplies an arginine finger into the active site.
00:52:23.04 You have Gln1 mutation--any mutation of glutamine 61 is an oncogene
00:52:28.29 because it misses, then, the catalytic residue (the system).
00:52:33.00 You have glycine mutants which are sterically compromised to do GTP hydrolysis.
00:52:38.21 There is no way that the arginine finger
00:52:42.06 can go into its proper position when there is a mutation of glycine 12.
00:52:46.04 I also showed you that there is a strongly bound GDP-Pi intermediate.
00:52:50.02 And that Pi release in the system becomes rate-limiting.
00:52:54.28 Whereas without GAP, the chemical cleavage reaction is the rate-limiting step.
00:53:00.23 The second conclusion--more general to the Ras superfamily.
00:53:05.01 Ras proteins are all incomplete enzymes.
00:53:07.20 They all hydrolyze GTP very, very slowly.
00:53:09.27 They have cognate GAPs.
00:53:12.02 So each of the sub-family proteins and sometimes even proteins within the sub-family,
00:53:16.24 have a specific cognate GAP that are required for fast GTP hydrolysis, for catalysis.
00:53:22.17 Some GAPs supply an arginine finger
00:53:25.02 and I have shown you now many different examples--Ras and Ran and Rho.
00:53:30.15 RabGAPs deliver an agrinine and a Gln.
00:53:34.23 Some GAPs supply an Asn thumb.
00:53:37.00 I showed you the example of RapGAP and Tuberin probably does the same thing.
00:53:41.03 Pi release is very often rate-limiting.
00:53:44.10 And what is even more important, and that is my message for the whole talk,
00:53:47.19 is that the perturbed GTPase reaction is involved in a number of diseases:
00:53:53.24 cancer, neurofibromatosis, tuberous sclerosis,
00:53:58.03 retinitis pigmentosa and many more that I haven't talked about.
00:54:01.07 Thank you for your attention. But I would.
00:54:03.00 Before I stop, let me first thank the people that have done the work.
00:54:07.18 The oldest story that I have told you about is that of Ras and RasGAP,
00:54:11.22 which was done by three post-docs in the lab, Reza Ahmadian, Klaus Scheffzek and Robert Mittal.
00:54:17.23 The RapGAP story was done by the students Oli Daumke, Astrid Kramer,
00:54:23.14 Partha Chakrabarti and Andrea Scrima.
00:54:26.18 And the RP2 story was done by Stefan Veltel and Karin Kuhnel.
00:54:32.04 And a lot of the movies that I have been showing you are doen by Ingrid Vetter
00:54:36.20 who also runs my crystallography lab.
00:54:38.15 And she was very, very helpful in almost all the projects.
00:54:42.13 And we have. on the FTIR we have collaborations with
00:54:46.03 our colleagues at the University of Bochum which is Klaus Gerwert and Carsten Kotting.
00:54:51.03 Thank you for your attention.

  • Part 1: GTP-Binding Proteins as Molecular Switches

Substrate Level Phosphorylation

The process of substrate-level phosphorylation is conceptually simple. In practice, however, it is complex because specific individual chemical reactions must be catalyzed by specific individual enzymes to produce ATP.

Figure legend: With substrate-level phosphorylation a specific enzyme, other than the ATP synthase involved in chemiosmosis, catalyzes the phosphorylation of ADP to generate ATP. There are two enzymes associated with glycolysis where such reactions take place &ndash phosphoglycerate kinase and pyruvate kinase &ndash and one associated with the Kreb's cycle, succinyl coenzyme A synthase. In the latter it is GTP rather than ATP that is immediately produced though the GTP is then converted to ATP.

Examples of substrate-level phosphorylation are the removal of inorganic phosphates from 1,3-biphosphoglycerate or phosphoenolpyruvate to form 3-phosphoglycerate or pyruvate, respectively, as well as ATP. Those substrate-level phosphorylation steps are seen in glycolysis, and substrate-level phosphorylation is also seen in the Kreb's citric acid cycle.

Substrate-level phosphorylation is simply an enzymatically catalyzed chemical reaction, albeit one that yields ATP . By contrast is phosphorylation that is effected via chemiosmotic processes such as what is described as oxidative phosphorylation. The latter is a process in which ATP production involves far more individual molecular players including an electron transport chain, the generation of a proton-motive force (which requires an intact lipid bilayer and associated cellular compartmentalization), and a complex enzyme known as ATP synthase.


Tubulin and microtubule-mediated processes, like cell locomotion, were seen by early microscopists, like Leeuwenhoek (1677). However, the fibrous nature of flagella and other structures were discovered two centuries later, with improved light microscopes, and confirmed in the 20th century with the electron microscope and biochemical studies. [8]

Microtubule in vitro assays for motor proteins such as dynein and kinesin are researched by fluorescently tagging a microtubule and fixing either the microtubule or motor proteins to a microscope slide then visualizing the slide with video-enhanced microscopy to record the travel of the microtubule motor proteins. This allows the movement of the motor proteins along the microtubule or the microtubule moving across the motor proteins. [9] Consequently, some microtubule processes can be determined by kymograph. [10]

In eukaryotes, microtubules are long, hollow cylinders made up of polymerised α- and β-tubulin dimers. [12] The inner space of the hollow microtubule cylinders is referred to as the lumen. The α and β-tubulin subunits are identical at the amino acid level, and each have a molecular weight of approximately 50 kDa. [13]

These α/β-tubulin dimers polymerize end-to-end into linear protofilaments that associate laterally to form a single microtubule, which can then be extended by the addition of more α/β-tubulin dimers. Typically, microtubules are formed by the parallel association of thirteen protofilaments, although microtubules composed of fewer or more protofilaments have been observed in various species [14] as well as in vitro. [15]

Microtubules have a distinct polarity that is critical for their biological function. Tubulin polymerizes end to end, with the β-subunits of one tubulin dimer contacting the α-subunits of the next dimer. Therefore, in a protofilament, one end will have the α-subunits exposed while the other end will have the β-subunits exposed. These ends are designated the (−) and (+) ends, respectively. The protofilaments bundle parallel to one another with the same polarity, so, in a microtubule, there is one end, the (+) end, with only β-subunits exposed, while the other end, the (−) end, has only α-subunits exposed. While microtubule elongation can occur at both the (+) and (−) ends, it is significantly more rapid at the (+) end. [16]

The lateral association of the protofilaments generates a pseudo-helical structure, with one turn of the helix containing 13 tubulin dimers, each from a different protofilament. In the most common "13-3" architecture, the 13th tubulin dimer interacts with the next tubulin dimer with a vertical offset of 3 tubulin monomers due to the helicity of the turn. There are other alternative architectures, such as 11-3, 12-3, 14-3, 15-4, or 16-4, that have been detected at a much lower occurrence. [17] Microtubules can also morph into other forms such as helical filaments, which are observed in protist organisms like foraminifera. [18] There are two distinct types of interactions that can occur between the subunits of lateral protofilaments within the microtubule called the A-type and B-type lattices. In the A-type lattice, the lateral associations of protofilaments occur between adjacent α and β-tubulin subunits (i.e. an α-tubulin subunit from one protofilament interacts with a β-tubulin subunit from an adjacent protofilament). In the B-type lattice, the α and β-tubulin subunits from one protofilament interact with the α and β-tubulin subunits from an adjacent protofilament, respectively. Experimental studies have shown that the B-type lattice is the primary arrangement within microtubules. However, in most microtubules there is a seam in which tubulin subunits interact α-β. [19]

Some species of Prosthecobacter also contain microtubules. The structure of these bacterial microtubules is similar to that of eukaryotic microtubules, consisting of a hollow tube of protofilaments assembled from heterodimers of bacterial tubulin A (BtubA) and bacterial tubulin B (BtubB). Both BtubA and BtubB share features of both α- and β-tubulin. Unlike eukaryotic microtubules, bacterial microtubules do not require chaperones to fold. [20] In contrast to the 13 protofilaments of eukaryotic microtubules, bacterial microtubules comprise only five. [21]

Microtubules are part of the cytoskeleton, a structural network within the cell's cytoplasm. The roles of the microtubule cytoskeleton include mechanical support, organization of the cytoplasm, transport, motility and chromosome segregation. In developing neurons microtubules are known as neurotubules, [22] and they can modulate the dynamics of actin, another component of the cytoskeleton. [23] A microtubule is capable of growing and shrinking in order to generate force, and there are motor proteins that allow organelles and other cellular components to be carried along a microtubule. This combination of roles makes microtubules important for organizing and moving intracellular constituents.

The organization of microtubules in the cell is cell-type specific. In epithelia, the minus-ends of the microtubule polymer are anchored near the site of cell-cell contact and organized along the apical-basal axis. After nucleation, the minus-ends are released and then re-anchored in the periphery by factors such as ninein and PLEKHA7. [24] In this manner, they can facilitate the transport of proteins, vesicles and organelles along the apical-basal axis of the cell. In fibroblasts and other mesenchymal cell-types, microtubules are anchored at the centrosome and radiate with their plus-ends outwards towards the cell periphery (as shown in the first figure). In these cells, the microtubules play important roles in cell migration. Moreover, the polarity of microtubules is acted upon by motor proteins, which organize many components of the cell, including the endoplasmic reticulum and the Golgi apparatus.

Nucleation Edit

Nucleation is the event that initiates the formation of microtubules from the tubulin dimer. Microtubules are typically nucleated and organized by organelles called microtubule-organizing centres (MTOCs). Contained within the MTOC is another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits of the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a lock washer-like structure known as the "γ-tubulin ring complex" (γ-TuRC). This complex acts as a template for α/β-tubulin dimers to begin polymerization it acts as a cap of the (−) end while microtubule growth continues away from the MTOC in the (+) direction. [25]

The centrosome is the primary MTOC of most cell types. However, microtubules can be nucleated from other sites as well. For example, cilia and flagella have MTOCs at their base termed basal bodies. In addition, work from the Kaverina group at Vanderbilt, as well as others, suggests that the Golgi apparatus can serve as an important platform for the nucleation of microtubules. [26] Because nucleation from the centrosome is inherently symmetrical, Golgi-associated microtubule nucleation may allow the cell to establish asymmetry in the microtubule network. In recent studies, the Vale group at UCSF identified the protein complex augmin as a critical factor for centrosome-dependent, spindle-based microtubule generation. It that has been shown to interact with γ-TuRC and increase microtubule density around the mitotic spindle origin. [27]

Some cell types, such as plant cells, do not contain well defined MTOCs. In these cells, microtubules are nucleated from discrete sites in the cytoplasm. Other cell types, such as trypanosomatid parasites, have a MTOC but it is permanently found at the base of a flagellum. Here, nucleation of microtubules for structural roles and for generation of the mitotic spindle is not from a canonical centriole-like MTOC.

Polymerization Edit

Following the initial nucleation event, tubulin monomers must be added to the growing polymer. The process of adding or removing monomers depends on the concentration of αβ-tubulin dimers in solution in relation to the critical concentration, which is the steady state concentration of dimers at which there is no longer any net assembly or disassembly at the end of the microtubule. If the dimer concentration is greater than the critical concentration, the microtubule will polymerize and grow. If the concentration is less than the critical concentration, the length of the microtubule will decrease. [28]

Dynamic instability Edit

Dynamic instability refers to the coexistence of assembly and disassembly at the ends of a microtubule. The microtubule can dynamically switch between growing and shrinking phases in this region. [29] Tubulin dimers can bind two molecules of GTP, one of which can be hydrolyzed subsequent to assembly. During polymerization, the tubulin dimers are in the GTP-bound state. [12] The GTP bound to α-tubulin is stable and it plays a structural function in this bound state. However, the GTP bound to β-tubulin may be hydrolyzed to GDP shortly after assembly. The assembly properties of GDP-tubulin are different from those of GTP-tubulin, as GDP-tubulin is more prone to depolymerization. [30] A GDP-bound tubulin subunit at the tip of a microtubule will tend to fall off, although a GDP-bound tubulin in the middle of a microtubule cannot spontaneously pop out of the polymer. Since tubulin adds onto the end of the microtubule in the GTP-bound state, a cap of GTP-bound tubulin is proposed to exist at the tip of the microtubule, protecting it from disassembly. When hydrolysis catches up to the tip of the microtubule, it begins a rapid depolymerization and shrinkage. This switch from growth to shrinking is called a catastrophe. GTP-bound tubulin can begin adding to the tip of the microtubule again, providing a new cap and protecting the microtubule from shrinking. This is referred to as "rescue". [31]

"Search and capture" model Edit

In 1986, Marc Kirschner and Tim Mitchison proposed that microtubules use their dynamic properties of growth and shrinkage at their plus ends to probe the three dimensional space of the cell. Plus ends that encounter kinetochores or sites of polarity become captured and no longer display growth or shrinkage. In contrast to normal dynamic microtubules, which have a half-life of 5–10 minutes, the captured microtubules can last for hours. This idea is commonly known as the "search and capture" model. [32] Indeed, work since then has largely validated this idea. At the kinetochore, a variety of complexes have been shown to capture microtubule (+)-ends. [33] Moreover, a (+)-end capping activity for interphase microtubules has also been described. [34] This later activity is mediated by formins, [35] the adenomatous polyposis coli protein, and EB1, [36] a protein that tracks along the growing plus ends of microtubules.

Post-translational modifications Edit

Although most microtubules have a half-life of 5–10 minutes, certain microtubules can remain stable for hours. [34] These stabilized microtubules accumulate post-translational modifications on their tubulin subunits by the action of microtubule-bound enzymes. [37] [38] However, once the microtubule depolymerizes, most of these modifications are rapidly reversed by soluble enzymes. Since most modification reactions are slow while their reverse reactions are rapid, modified tubulin is only detected on long-lived stable microtubules. Most of these modifications occur on the C-terminal region of alpha-tubulin. This region, which is rich in negatively charged glutamate, forms relatively unstructured tails that project out from the microtubule and form contacts with motors. Thus, it is believed that tubulin modifications regulate the interaction of motors with the microtubule. Since these stable modified microtubules are typically oriented towards the site of cell polarity in interphase cells, this subset of modified microtubules provide a specialized route that helps deliver vesicles to these polarized zones. These modifications include:

    : the removal of the C-terminal tyrosine from alpha-tubulin. This reaction exposes a glutamate at the new C-terminus. As a result, microtubules that accumulate this modification are often referred to as Glu-microtubules. Although the tubulin carboxypeptidase has yet to be identified, the tubulin—tyrosine ligase (TTL) is known. [39]
  • Delta2: the removal of the last two residues from the C-terminus of alpha-tubulin. [40] Unlike detyrosination, this reaction is thought to be irreversible and has only been documented in neurons. : the addition of an acetyl group to lysine 40 of alpha-tubulin. This modification occurs on a lysine that is accessible only from the inside of the microtubule, and it remains unclear how enzymes access the lysine residue. The nature of the tubulin acetyltransferase remains controversial, but it has been found that in mammals the major acetyltransferase is ATAT1. [41] however, the reverse reaction is known to be catalyzed by HDAC6. [42] : the addition of a glutamate polymer (typically 4-6 residues long [43] ) to the gamma-carboxyl group of any one of five glutamates found near the end of alpha-tubulin. Enzymes related to TTL add the initial branching glutamate (TTL4,5 and 7), while other enzymes that belong to the same family lengthen the polyglutamate chain (TTL6,11 and 13). [38] : the addition of a glycine polymer (2-10 residues long) to the gamma-carboxyl group of any one of five glutamates found near the end of beta-tubulin. TTL3 and 8 add the initial branching glycine, while TTL10 lengthens the polyglycine chain. [38]

Tubulin-binding drugs and chemical effects Edit

A wide variety of drugs are able to bind to tubulin and modify its assembly properties. These drugs can have an effect at intracellular concentrations much lower than that of tubulin. This interference with microtubule dynamics can have the effect of stopping a cell's cell cycle and can lead to programmed cell death or apoptosis. However, there are data to suggest that interference of microtubule dynamics is insufficient to block the cells undergoing mitosis. [44] These studies have demonstrated that suppression of dynamics occurs at concentrations lower than those needed to block mitosis. Suppression of microtubule dynamics by tubulin mutations or by drug treatment have been shown to inhibit cell migration. [45] Both microtubule stabilizers and destabilizers can suppress microtubule dynamics.

The drugs that can alter microtubule dynamics include:

  • The cancer-fighting taxane class of drugs (paclitaxel (taxol) and docetaxel) block dynamic instability by stabilizing GDP-bound tubulin in the microtubule. Thus, even when hydrolysis of GTP reaches the tip of the microtubule, there is no depolymerization and the microtubule does not shrink back.

Taxanes (alone or in combination with platinum derivatives (carboplatine) or gemcitabine) are used against breast and gynecological malignancies, squamous-cell carcinomas (head-and-neck cancers, some lung cancers), etc.

  • The epothilones, e.g. Ixabepilone, work in a similar way to the taxanes.
  • Vinorelbine, Nocodazole, vincristine, and colchicine have the opposite effect, blocking the polymerization of tubulin into microtubules. binds to the (+) growing end of the microtubules. Eribulin exerts its anticancer effects by triggering apoptosis of cancer cells following prolonged and irreversible mitotic blockade.

Expression of β3-tubulin has been reported to alter cellular responses to drug-induced suppression of microtubule dynamics. In general the dynamics are normally suppressed by low, subtoxic concentrations of microtubule drugs that also inhibit cell migration. However, incorporating β3-tubulin into microtubules increases the concentration of drug that is needed to suppress dynamics and inhibit cell migration. Thus, tumors that express β3-tubulin are not only resistant to the cytotoxic effects of microtubule targeted drugs, but also to their ability to suppress tumor metastasis. Moreover, expression of β3-tubulin also counteracts the ability of these drugs to inhibit angiogenesis which is normally another important facet of their action. [ citation needed ]

Microtubule polymers are extremely sensitive to various environmental effects. Very low levels of free calcium can destabilize microtubules and this prevented early researchers from studying the polymer in vitro. [12] Cold temperatures also cause rapid depolymerization of microtubules. In contrast, heavy water promotes microtubule polymer stability. [46]

Microtubule-associated proteins (MAPs) Edit

MAPs have been shown to play a crucial role in the regulation of microtubule dynamics in-vivo. The rates of microtubule polymerization, depolymerization, and catastrophe vary depending on which microtubule-associated proteins (MAPs) are present. The originally identified MAPs from brain tissue can be classified into two groups based on their molecular weight. This first class comprises MAPs with a molecular weight below 55-62 kDa, and are called τ (tau) proteins. In-vitro, tau proteins have been shown to directly bind microtubules, promote nucleation and prevent disassembly, and to induce the formation of parallel arrays. [47] Additionally, tau proteins have also been shown to stabilize microtubules in axons and have been implicated in Alzheimer's disease. [48] The second class is composed of MAPs with a molecular weight of 200-1000 kDa, of which there are four known types: MAP-1, MAP-2, MAP-3 and MAP-4. MAP-1 proteins consists of a set of three different proteins: A, B and C. The C protein plays an important role in the retrograde transport of vesicles and is also known as cytoplasmic dynein. MAP-2 proteins are located in the dendrites and in the body of neurons, where they bind with other cytoskeletal filaments. The MAP-4 proteins are found in the majority of cells and stabilize microtubules. In addition to MAPs that have a stabilizing effect on microtubule structure, other MAPs can have a destabilizing effect either by cleaving or by inducing depolymerization of microtubules. Three proteins called katanin, spastin, and fidgetin have been observed to regulate the number and length of microtubules via their destabilizing activities. Furthermore, KIAA1211L is predicted to be localized to the microtubules. [49]

Plus-end tracking proteins (+TIPs) Edit

Plus end tracking proteins are MAP proteins which bind to the tips of growing microtubules and play an important role in regulating microtubule dynamics. For example, +TIPs have been observed to participate in the interactions of microtubules with chromosomes during mitosis. The first MAP to be identified as a +TIP was CLIP170 (cytoplasmic linker protein), which has been shown to play a role in microtubule depolymerization rescue events. Additional examples of +TIPs include EB1, EB2, EB3, p150Glued, Dynamitin, Lis1, CLIP115, CLASP1, and CLASP2. [ citation needed ]


Transcription is the first part of the central dogma of molecular biology: DNA &rarr RNA. It is the transfer of genetic instructions in DNA to mRNA. Transcription happens in the nucleus of the cell. During transcription, a strand of mRNA is made that is complementary to a strand of DNA called a gene. A gene can easily be identified from the DNA sequence. A gene contains the basic three regions, promoter, coding sequence (reading frame), and terminator. There are more parts of a gene which are illustrated in Figure (PageIndex<3>).

Figure (PageIndex<3>): The major components of a gene. 1. promoter, 2. transcription initiation, 3. 5' upstream untranslated region, 4. translation start codon site, 5. protein-coding sequence, 6. translation stop codon region, 7. 3' downstream untranslated region, and 8. terminator.

Steps of Transcription

Transcription takes place in three steps, called initiation, elongation, and termination. The steps are illustrated in Figure (PageIndex<4>).

  1. Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can &ldquoread&rdquo the bases in one of the DNA strands. The enzyme is ready to make a strand of mRNA with a complementary sequence of bases. The promoter is not part of the resulting mRNA
  2. Elongation is the addition of nucleotides to the mRNA strand.
  3. Termination is the ending of transcription. As RNA polymerase transcribes the terminator, it detaches from DNA. The mRNA strand is complete after this step. Figure (PageIndex<4>): Transcription occurs in the three steps - initiation, elongation, and termination

Processing mRNA

In eukaryotes, the new mRNA is not yet ready for translation. At this stage, it is called pre-mRNA, and it must go through more processing before it leaves the nucleus as mature mRNA. The processing may include the addition of a 5' cap, splicing, editing, and 3' polyadenylation (poly-A) tail. These processes modify the mRNA in various ways. Such modifications allow a single gene to be used to make more than one protein. See Figure (PageIndex<5>) as you read below:

  • 5' cap protects mRNA in the cytoplasm and helps in the attachment of mRNA with the ribosome for translation.
  • Splicing removes introns from the protein-coding sequence of mRNA. Introns are regions that do not code for the protein. The remaining mRNA consists only of regions called exons that do code for the protein.
  • Editing changes some of the nucleotides in mRNA. For example, a human protein called APOB, which helps transport lipids in the blood, has two different forms because of editing. One form is smaller than the other because editing adds an earlier stop signal in mRNA.
  • Polyadenylation adds a &ldquotail&rdquo to the mRNA. The tail consists of a string of As (adenine bases). It signals the end of mRNA. It is also involved in exporting mRNA from the nucleus, and it protects mRNA from enzymes that might break it down.

What are the features of ATP which lead it to being termed the 'universal currency' of the cell?

Adenosine triphosphate (ATP for short) is used to provide the energy for many cellular functions - by both animal and plant cells - as cells are unable to source their energy directly from sugars such as glucose. ATP is formed during respiration, when the energy released from ingested glucose powers the addition of a inorganic phosphate molecule to a molecule of adenosine diphosphate (ADP), the enzyme ATP synthase is responsible for this. ATP itself is a small, soluble molecule which can be easily broken down and transported around the cell. The many tasks ATP is used for include the transport work of moving substances, such as ions, across cell membranes in active transport and within the processes of muscular contraction and cellular growth and division too. As well as this, ATP is readily available within cells upon demand as stores are released in manageable amounts, meaning there is no wasted energy. The energy-containing phosphate bond is easily broken down to release the energy it holds and to further this ATP molecules are not lipid soluble and therefore they are unable to pass beyond the cell membrane, ensuring the cell always always has an immediate energy source. Finally, ATP can be deemed a good energy source as it has the ability to transfer a phosphate group, and therefore energy, to other molecules.

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Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.

Outcomes of Glycolysis

One glucose molecule produces four ATP, two NADH, and two pyruvate molecules during glycolysis.

Learning Objectives

Describe the energy obtained from one molecule of glucose going through glycolysis

Key Takeaways

Key Points

  • Although four ATP molecules are produced in the second half, the net gain of glycolysis is only two ATP because two ATP molecules are used in the first half of glycolysis.
  • Enzymes that catalyze the reactions that produce ATP are rate-limiting steps of glycolysis and must be present in sufficient quantities for glycolysis to complete the production of four ATP, two NADH, and two pyruvate molecules for each glucose molecule that enters the pathway.
  • Red blood cells require glycolysis as their sole source of ATP in order to survive, because they do not have mitochondria.
  • Cancer cells and stem cells also use glycolysis as the main source of ATP (process known as aerobic glycolysis, or Warburg effect).

Key Terms

  • pyruvate: any salt or ester of pyruvic acid the end product of glycolysis before entering the TCA cycle

Outcomes of Glycolysis

Glycolysis starts with one molecule of glucose and ends with two pyruvate (pyruvic acid) molecules, a total of four ATP molecules, and two molecules of NADH. Two ATP molecules were used in the first half of the pathway to prepare the six-carbon ring for cleavage, so the cell has a net gain of two ATP molecules and 2 NADH molecules for its use. If the cell cannot catabolize the pyruvate molecules further (via the citric acid cycle or Krebs cycle), it will harvest only two ATP molecules from one molecule of glucose.

Glycolysis produces 2 ATP, 2 NADH, and 2 pyruvate molecules: Glycolysis, or the aerobic catabolic breakdown of glucose, produces energy in the form of ATP, NADH, and pyruvate, which itself enters the citric acid cycle to produce more energy.

Mature mammalian red blood cells do not have mitochondria and are not capable of aerobic respiration, the process in which organisms convert energy in the presence of oxygen. Instead, glycolysis is their sole source of ATP. Therefore, if glycolysis is interrupted, the red blood cells lose their ability to maintain their sodium-potassium pumps, which require ATP to function, and eventually, they die. For example, since the second half of glycolysis (which produces the energy molecules) slows or stops in the absence of NAD+, when NAD+ is unavailable, red blood cells will be unable to produce a sufficient amount of ATP in order to survive.

Additionally, the last step in glycolysis will not occur if pyruvate kinase, the enzyme that catalyzes the formation of pyruvate, is not available in sufficient quantities. In this situation, the entire glycolysis pathway will continue to proceed, but only two ATP molecules will be made in the second half (instead of the usual four ATP molecules). Thus, pyruvate kinase is a rate-limiting enzyme for glycolysis.

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