Effective molarity

Transcription

In this webcast what we want to do is take a look at the ways that enzymes bring about stability of the transition state relative to the substrate. There's basically two most important factors, one is known as proximity, also sometimes called effective molarity, and the second factor is known as orientation effects. Let's take a look at the effects of proximity as illustrated by the first two reactions that are shown here. Both of these are hydrolysis of these phenyl esters. In the first case, the catalyst trimethylamine is delivered intermolecularly. The reaction is going to be run at some fixed concentration of this trimethylamino group so that it's run under pseudo first-order conditions, and that'll allow us to compare the relative rates of that reaction to the rate of the reaction in which the amino group is bound covalently, it's tethered to the substrate four atoms away. In the case of the second reaction, the hydrolysis proceeds a million times faster, more than a million times faster than it does in the first case. How is it that this trialkylamino group catalyzes the reaction? And what's the effect of tethering that group to the carbonyl? Well we could imagine one of two possible reaction pathways that this amino group could catalyze the process. It could be a nucleophilic catalyst, and so for example in the case of the second reaction, we're going to benefit from the close proximity of that nucleophile to the carbonyl to make this intermediate that's shown here. That's going to go on and do a β-elimination, kick out the phenoxide group. That'll generate an intermediate that I'm drawing for you, and it's really a very good leaving group that will subsequently undergo hydrolysis. It's a good leaving group because it's an acyl ammonium and so the positive charge makes this a very good leaving group, that carbonyl susceptible to attack by water and then the ammonium group will be expelled. So, again, ah, the reason that this is going to be so enhanced in the second case is because of the proximity, that amino group is always going to be located in a very short distance away from the carbonyl. The second case is a general base catalyzed process. And once again, as you can see, the effective molarity in the vicinity of the carbonyl is always going to be very high when the molecule- the dimethylamino group is tethered to that carbonyl group. So it can serve as a base, in this case you can see it’s serving as a general base catalyzed mechanism. And it’s always going to have this high effective molarity, due to the proximity. Enzymes do something very similar. They use the scaffold of that entire protein to position side chains just in the right spot next to the substrate to achieve high effective molarity when the enzyme binds its substrate. So that’s the concept of proximity. Let’s take a look at the orientation factor next. We’re going to illustrate this for a specific reaction, this is a ah, phosphoralation reaction. This is the guanadinium group that’s going to pick up a phosphate group. And this is actually a crystal structure that’s been inhibited with nitrate. Now normally the enzyme’s natural substrate would be ATP and it’s basically a phosphate transfer. The idea of orientation is simply that there’s some level of orbital sphering going on and that’s the thing that I want you to pick up on. Not so much the details of this reaction, we’ll talk about the details of the reaction a little bit later on. But basically in this reaction, what’s going to go on is the key step involves an n to σ* interaction and so the bond that’s going to break, the σ* bond, is going to be this bond right here. That σ* is perfectly aligned in the enzyme substrate complex with the nonbonding lone pair. That σ* is perfectly aligned so that there is the overlap- maximum overlap in the σ type mode between that nonbonding pair and σ*. Both the effects of proximity and orientation can really be broken down in the statement that’s summarized here. Basically the secret to the power of enzyme catalysis is that was- what once was an intermolecular reaction, is now really effectively by the enzyme substrate complex, an intramolecular reaction that benefits from these effects of proximity and orientation.