EPSP synthase

Transcription

Remembering that phosphorylation is nature's way of transforming a hydroxyl group into a good leaving group, you can find a couple of examples of this strategy in the shikimate pathway. I won't spend much time on this one. I've already talked about it just a little bit, but the chorismate synthase is this 1,4 addition where the phosphate group is lost to introduce a new degree of unsaturation into the six-membered ring. The second, and much more interesting example, is the substitution reaction that's shown here. So we're going to do a substitution reaction on the enol, phosphoenol- pyruvate sp2 carbon, so we're going to take and at that sp2 center, replace the phosphate group with the hydroxyl group on this shikimate acid. And so how does that take place? Well, that mec- mechanism as we'll see takes place by an additional elimination process. Actually, you should by now be used to thinking about substitution at sp2 centers as taking place by this mechanism, there'll be a key tetrahedral intermediate that gets involved. It's all catalyzed by EPSP synthase, enolpyruvate shikimate phosphate synthase, and this is the step where the inhibitor that's in Roundup gets involved and basically blocks the, ah, this addition elimination, ah, mechanism from taking place. So let's understand the mechanism so we can understand how Roundup, a competitive inhibitor, that's a transition state analog of a key portion of the rate- the rate determining step of this, ah, substitution reaction - how does it do it's interference, how does it cause it's, ah, interference? Well, it actually is all in the first step that I'm going to show you where, as we're going to get set up to do that addition elimination, the first thing that happens is this double bond of the phosphoenolpyruvate, which benefits from donation of electron density from the nonbonding pair of electrons into π*, makes that a fairly nucleophilic carbon-carbon double bond. And, and so, the enzyme active site has a side chain that delivers a proton to that, and we can think about this step as an electrophile association step, and in fact, that's how we want to understand it in terms of trying to understand how the inhibitor Roundup gets involved. This is in fact the rate determining step for the overall substitution process. And if I draw it as this, without first invoking that lone pair, but I just think about this as an electrophile association step, which of course as you know will make a carbocation, we see that the structure of the, ah, intermediate is going to have a partial positive charge at the position, ah, that, that's shown there. That's key because we want to mimic that in our transition state analog, the phosph-, the gly- glyphos that's, ah, the inhibitor that is present in Roundup. Well, there's obviously another important resonance contributor, that's the, ah, N to A donation that we see there that puts the, ah, carbon- oxygen double bond that's going to be a very good electrophile for the first step, or the second step of this process. This is actually the addition step, so up till now, we've just been getting ready to do the addition step of the addition elimination substitution reaction. So let's see that, how that unfolds. Here we have, ah, the shikimate acid and it's going to be this hydroxyl group, which is going to serve as the nucleophile. It's going to be general base-catalyzed, and, and so the addition step looks like this. It's a general-base catalyzed ADN step that's going to make a new oxygen, ah, carbon bond and the structure of that is shown over here. We've now protonated the conjugate, ah, the base, as, as its conjugate acid and so it can serve as a general acid in doing an elimination step. So, our, ah, what was functioning as the acid is now positioned as a base and that's going to be used to eliminate one of the carbon- hydrogen bonds, we'll kick out the good leaving group that's going to be general acid catalyzed, and so the final substitution reaction, which we needed to return that carbon back to its sp2 form, that ah where the substitution reaction takes place, gives us that structure. Alright, now we want to understand how, ah, the inhibitor glyphos mimics the rate determining step, which is the generation of this intermediate that is set up to do the addition process. So here's the overall step. Basically, it's the developing positive charge here, and we can see that in the transition state, um, there's, ah, that is, um, in this, for the addition step that there's a partial positive charge on that carbon. It has a geometry that's beginning to take a tetrahedral geometry and that's the, that is what we're trying to mimic in the inhibitor. We want to fit a structure in that will basically be completely stable, but block the active site by mimicking the electronic structure, the partial positive charge, and the geometry as being tetrahedral. And that's the enzyme inhibitor; phosphonomethyl glycine is the formal name. Ah, most people who use this call it glyphos. It's a carbocation mimic. It's, ah, basically going to have the geometry and electronic features of this, ah, transition state structure here. We can see that by doing our superposition idea, and you can see how phosphonomethyl glycine overlays very nicely on top of this transition structure that we drew. I was, ah, I grew up on a farm and I spent, ah, a, many summers walking soy beans because they needed to be weeded. Um, sometime in the early 1990s there was the introduction of a new breed of soybeans, Roundup ready soybeans. This is a wonderful marriage of genetics and molecular biology with chemistry because this was a genetic mutant of the EPSP synthase enzyme which was tolerant to glycine, ah, this ah, to, to, to the, the phosphonomethyl glycine and, ah, and so it's really a remarkable feat in that we have soybeans that basically have a mutated enzyme which can um basically survive in the presence of Roundup so that one can spray the soybean field and you now see these spectacularly clean soybean fields today. People don't have to do the suffering of the summer heat that I had to experience when I was walking soybeans as a, as a, ah, as a teenager.