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  • ELIZABETH NOLAN: So last time, we

  • were talking about these aminoacyl tRNA synthetases that

  • are responsible for attaching amino acid

  • monomers to the three prime end of tRNAs.

  • And we were looking at the isoleucyl aminoacyl tRNA

  • synthetase as an example, looking at experiments that

  • were done to study mechanisms.

  • So recall, we left off having discussed a two-step model,

  • where there's an intermediate, an amino adenylate formed.

  • And then, in the second step, there's

  • transfer of that amino acid to the tRNA by the aaRS.

  • And so we looked at some data from steady-state kinetic

  • experiments.

  • Recall that a C14 radiolabel was used to watch transfer,

  • and then we closed discussing an ATP-PPi exchange assay which

  • gave evidence for formation of that amino adenylate

  • intermediate.

  • Right?

  • And then, lastly, we talked about use of a stopped-flow

  • to do experiments that allow you to look at early points

  • within a reaction.

  • And so what we're going to do is to close these discussions

  • of experiments and this aaRS mechanism

  • is just look at one more experiment that

  • was done to further probe the rate-determining step

  • of this reaction using the stopped-flow.

  • OK?

  • And so this experiment pertains more to reaction kinetics,

  • and the question is, let's monitor

  • transfer of the amino acid to the tRNA

  • by another method here.

  • These experiments were set up in two different ways

  • depending on what components were mixed.

  • And if you just rewind to Monday and recall the ATP-PPi exchange

  • assay and the steps in that assay, in that we showed

  • that the amino adenylate intermediate remained

  • bound to the enzyme there.

  • Recall then only PPi was released in that assay.

  • And so in these experiments, the fact

  • that the amino adenylate can remain bound

  • was taken advantage of.

  • And the researchers were actually

  • able to have a preformed complex there, so basically

  • starting after step two.

  • So in experiment one, how I'm going to show

  • these is by drawing the two syringes

  • and listing the components of each syringe.

  • And this is a good way for setting up problems

  • within the problem sets, thinking

  • about stopped-flow experiments.

  • So the question is what are we going to mix?

  • So we have syringe one and syringe two,

  • and recall that these go to some mixer.

  • So the two solutions can be rapidly mixed,

  • and that's where the chemistry is going to happen.

  • So in experiment one, in syringe one,

  • what we have is the purified complex.

  • OK?

  • So we have C-14 labeled isoleucine-AMP

  • bound to the aminoacyl tRNA synthetase

  • of a purified complex, here.

  • And then in this other syringe two, what we have is the tRNA.

  • OK?

  • So imagine these are rapidly mixed.

  • There'll be transfer of the radiolabeled isoleucine

  • to the tRNA, and so formation of that aminoacyl tRNA

  • can be monitored.

  • OK?

  • In the second experiment, we have just theme in variation,

  • and if you're interested in more details,

  • the reference is provided in the slides.

  • So again, in syringe two, we have the tRNA,

  • and in syringe one, what will be combined

  • are the components here.

  • OK?

  • So then, the question is, in each case, what do we see?

  • And those data are presented here from the paper,

  • and there's some additional details

  • about the experimental setup.

  • So effectively, what we're looking at on the y-axis

  • is the amount of tRNA that's been modified.

  • So tRNA acylation measured by transfer

  • of the radiolabel versus time.

  • And in the black circles, we have the data

  • from experiment one, shown here, and in the open circles,

  • we have the data from experiment two.

  • So what is the conclusion from these data?

  • And this value here is not similar to something we've

  • seen before in this system.

  • Both experimental setups are giving the same result. Right?

  • Effectively, these data are superimposable,

  • and they can be fit the same.

  • So what does that tell us about the rate-determining step?

  • AUDIENCE: [INAUDIBLE] versus forming the intermediate.

  • ELIZABETH NOLAN: Yeah.

  • Right.

  • Aminoacylation of tRNA is the rate-determining step.

  • So some of you suggested that in class on Monday.

  • Right?

  • So that's the case here.

  • OK?

  • So formation of the intermediate is much more rapid

  • than acylation of the tRNA here.

  • So we've examined now the mechanism

  • in terms of getting the amino acid onto the tRNA.

  • What do we need to think about next here?

  • So what we need to think about is fidelity.

  • OK, and we've looked at the overall rate of error

  • in protein biosynthesis, how often errors occur

  • on the order of 10 to the 3.

  • So how is the correct amino acid loaded onto the correct tRNA?

  • Each tRNA has an anticodon that is a cognate pair with a codon.

  • And so different tRNAs need to have

  • different amino acids attached.

  • OK, and what does that mean?

  • That means, in general, there's a dedicated aminoacyl tRNA

  • synthetase for each amino acid, in general here.

  • So how are amino acids with similar side chains

  • differentiated by these enzymes?

  • And is it possible for an incorrect amino acid

  • to get loaded onto a tRNA?

  • And if that happens, what are the consequences?

  • So we're going to examine fidelity some here.

  • And as background, an observation made,

  • say from studies like that ATP-PPi exchange assay,

  • is that some aminoacyl tRNA synthetases can activate

  • multiple amino acids, so not only the one

  • they're supposed to activate but also others.

  • So what does that mean?

  • That means that the enzyme can bind

  • and activate effectively the wrong amino acid,

  • and if we think about fidelity, we

  • can think about this as being a problem here.

  • So what happens?

  • What happens is that these enzymes have an editing

  • function, and they're able to sense if a wrong amino acid is

  • activated.

  • And then they have a way to deal with it,

  • and this is by hydrolysis.

  • OK?

  • And so let's consider an example, for instance, just

  • similar side chains.

  • So if we just consider, for instance, valine, isoleucine,

  • and threonine, these will be the players for our discussion.

  • OK?

  • They're different, but they're not too different.

  • Right?

  • Oops, sorry about this.

  • We're missing a methyl.

  • Valine, an isoleucine, we have a difference of a methyl group.

  • Threonine, we have this OH group.

  • Right?

  • And we can just ask the question,

  • for instance, how is valine differentiated from isoleucine

  • or threonine here?

  • And so as an example, what's found

  • is, if we consider our friend that we studied

  • for the mechanism here, what we find

  • is that this binds and activates isoleucine, as we saw,

  • but it will also bind and activate valine here.

  • And effectively, if this happens,

  • we have a mismatch, because the end result

  • will be isoleucine-RS with valine AMP bound here.

  • OK?

  • And what's found is that the catalytic efficiency or Kcat

  • over Km, in this case, is about 150-fold

  • less than the native substrate.

  • So that doesn't account for the 10 to the 3 error rate here.

  • So we need more specificity.

  • So what's going on?

  • So we're going to consider this editing function and a model

  • that's often used to describe how these aaRS do

  • editing is one of two sieves.

  • These enzymes don't actually have a sieve.

  • It's just a conceptual way to think about it.

  • So this double-sieve editing model

  • involves a first sieve which is considered to be a course one.

  • So imagine if you have like a change sorter.

  • It will let the quarters through as well as

  • the and dimes and the pennies.

  • There's some sort of discrimination

  • of amino acids based on size, and then

  • depending what gets through this first sieve or gate,

  • there's a second sieve which is considered to be a fine one.

  • And this one can differentiate perhaps on the basis of size

  • or maybe on hydrophilicity or hydrophobic of the side chain.

  • So effectively, if an incorrect amino acid passes through this

  • first sieve-- so in other words, if it binds to the enzyme

  • and becomes activated--

  • hydrolytic editing will occur.

  • OK?

  • So think about hydrolysis in terms of having

  • breakdown of these species.

  • So if the incorrect amino acid passes through

  • and is adenylated, there'll be hydrolysis.

  • So let's consider some examples so the first example here we

  • can consider this guy and isoleucine and valine.

  • So as I mentioned, this aaRS will activate both.

  • So in this case, the first sieve can't differentiate isoleucine

  • from valine.

  • They have similar sizes according to this aaRS.

  • But then what happens here in the second sieve,

  • isoleucine is too big, and so there's no hydrolysis,

  • and it moves on to form the desired charged tRNA.

  • In contrast, valine's a bit smaller.

  • It passes through the sieve, and it ends up being hydrolyzed.

  • So these aaRS also have an editing domain,

  • and this editing domain, as we'll

  • see in a few slides in a structure,

  • is responsible for this hydrolysis, so stated here.

  • Right?

  • Different sites, so there's an aminoacylation site

  • and an editing site here.

  • So valine can reach the editing site, but isoleucine cannot.

  • So how do you predict?

  • Just to keep in mind, every enzyme

  • is different in terms of the model

  • for discrimination and also when editing occurs.

  • So you really need to look at the data

  • when the data is presented to you to sort out how this works.

  • Let's just look at another example

  • with a cartoon depiction.

  • So this is for the valine RS, and we're

  • going to consider the three amino acids here--

  • valine, threonine, and isoleucine.

  • So in green, we have the first sieve,

  • and this is based on size.

  • So what do we see in this cartoon?

  • So threonine and valine make it through,

  • but isoleucine does not.

  • It's rejected right away, so it's never activated.

  • So if threonine and valine pass through, what happens?

  • We see each one is activated as the amino adenylate, and then

  • what?

  • Well, valine, we want to transfer the valine

  • to the tRNA, so it can move on and help

  • with protein synthesis.

  • If threonine's activated, and here we

  • see that threonine is transferred to the tRNA

  • as well, this is hydrolyzed by the editing site, in this case.

  • So the threonine is removed from the tRNA

  • with the anticodon for valine.

  • Right, so think about the ester bonds

  • that we saw last time in terms of the three prime end

  • of the tRNA being modified and the chemistry that

  • will happen there to result in hydrolysis of and release

  • of the amino acid here.

  • So what that cartoon hints to is that the hydrolysis can

  • occur at different steps.