Enzyme Electric Fields

How the heck do enzymes work, anyway? This question has been asked - often a bit more elegantly - for over a century now, and we're still working out the answer. There's no doubt that the active site of an enzyme is a very strange place, with interactions pushing and pulling on the substrate in ways that it would never experience out in bulk solvent. Specifically, an active site would be expected to stabilize the transition state of a reaction more than it stabilizes the starting material itself, thus lowering the energy barrier to the desired reaction. (And certainly more than it stabilizes the product - you actually want the product to fall out of the active site as quickly as possible so that another catalytic transformation can queue up).

Here's a new paper from a team at Stanford (earlier ChemrXiv version) that illustrates that with an ingenious probe technique. In a reaction that involves substantial development of charge in that transition state, you'd look for a complementary electrostatic environment in the enzyme's active site. But how to measure that? This paper uses liver alcohol dehydrogenase as the test enzyme, and N-cyclohexylformamide as the substrate. If you deuterate the aldehyde in that molecule, you end up with two groups (the C-O double bond and the C-D single bond) that are both very sensitive to the electric field around them, as measure by vibrational spectroscopy. And since these bonds point about 120 degrees from each other, you're getting two directional readouts at once. 

The authors measured effects on the vibrational spectra in various solvents and then in the presence of the enzyme. In solvents, they found that C=O bond frequency and the C-D one shifted in opposite directions as solvent polarity increased, which shows a stabilization of the former and a destabilization of the latter. The C=O is the big charge dipole in this system, as many of you will remember fondly from your organic chemistry, and it looks like the solvent organizes to stabilize that dipole at the expense of making the C-D part less stable. Interestingly, this effect was pretty similar across different solvents, suggesting that this stabilization/destabilization differs in degree but not in kind across different solvent polarities. They're all doing roughly the same thing: the C-D change was basically equivalent to the C=O change. The biggest shift was seen in water, which most of the time is about the most polar solvent you can get.

In the enzyme's active site, things got interesting. The shift of the two bonds was greater than even the water values, for one thing, the biggest stabilization/destabilization effect of them all. But at the same time, the linewidths of these two peaks were narrower than in any of the solvents, probably reflecting the smaller amount of fluctuations possible when the molecule was restrained in the active site, as opposed to floating around in solvent. The active site of this enzyme is pretty well mapped out, and the oxygen of the carbonyl is known to interact with a serine and a zinc atom. These are surely the source of the electric field difference noted experimentally. Meanwhile, the C-D (well, C-H in the normal substrate) doesn't really seem to be interacting with anything at all. 

But as mentioned above, this technique also gives you orientation information. This was really consistent for both bonds across the solvent measurements, indicating again that the solvents are all stabilizing the C=O in the same way (the electric field was just a few degrees off parallel to the C=O dipole). But the enzyme active site that angle is changed by about 14 degrees, in a direction not seen in any of the solvent measurements. That takes it from about seven degrees on one side of the C=O dipole to about seven degrees on the other, which sounds like you'd end up in the same general place, but the enzyme orientation actually puts more positive charge along the C-D bond and further destabilizes it. So you see a distinctive signature that leads to that larger-than-any-solvent result in the overall measurements.

And it supports the idea that enzyme active sites are ready with a pre-organized electrostatic environment, one that has surely been determined by evolutionary fitness over the years that gradually nudge things toward more and more catalytic efficiency. It's weird in there, and nothing like bulk solution. Being able to calculate the binding interactions in that weird environment and to compare them to the situation in bulk solvent is the fundamental problem in doing "virtual screening", which would (in its ideal state) allow us to dispense with physical target screening entirely and allow us to compute our way to binding-constant happiness. But to do that, we're going to have to know a lot more about the interactions of small molecules with protein surfaces, and this is the sort of work that is needed to keep shoving us along in that direction.

from Hacker News https://ift.tt/YJhyPmL