Nature of Enzymes

[Fortissimo]

Guys I've searched and searched but I havent found what I've been looking for.

What are the individual roles of the different amino acids found in enzymes, i.e. how do they aid catalysis. I know the basic Nitrogen of histidine is used to abstract a proton from serine, threonine or cysteine to activate it as a nucleophile but I dont even understand how that helps in catalysis! Not to talk of the other 19 amino acids. I need help people. Thanks.

[Mokele]

Well, amino acids can generally be sorted into 4 categories - non-polar, polar, acidic and basic. The problem is that one AA doesn't necessarily differ much from another with the same properties, so if you substitute, the enzyme will still function. So if you're looking for why one AA and not a similar one is used, the answer is mostly just luck.

 

However, the whole enzyme has multiple regions that do different things, like bind to the substrate or attach to another enzyme, in addition to the region that does what you're studying. Those different regions need different properties, so, for instance, the active site may need all acidic AAs, but the portion bound to the cell membrane (if that's the case) would have all non-polar AAs.

 

Mokele

[Kaeroll]

Guys I've searched and searched but I havent found what I've been looking for.

What are the individual roles of the different amino acids found in enzymes, i.e. how do they aid catalysis. I know the basic Nitrogen of histidine is used to abstract a proton from serine, threonine or cysteine to activate it as a nucleophile but I dont even understand how that helps in catalysis! Not to talk of the other 19 amino acids. I need help people. Thanks.

To be blunt - take a course on enzyme chemistry, preferably as given by a chemist. In the 2nd/3rd/4th years of my undergraduate chemistry degree, modules on the 'organic chemistry approach' to metabolism and enzyme chemistry were given, and answer - to some extent - your question. If this isn't an option, go to a university library and get out a decent biochemistry text. I find Berg to be thorough yet accessible.

 

Kaeroll

[ennui]

All of the amino acids will contribute to catalysis (the main function of an enzyme) either directly or indirectly. This is because the polypeptide chain undergoes a conformational change, and needs to have very specific biophysical constraints. In a lot of enzymes, even one mutation can lead to a loss or gain of function resulting in disease.

 

This isn't to say that all amino acids are directly in the active site. But it means that the whole shape of an enzymatic protein is important for function. The active site contains the catalytic portion of the enzyme.

 

As someone previously said, you can divide amino acids into groups. This might mean that if you have a mutation resulting in phenylalanine to tyrosine, you might be lucky and the enzyme could still work. This is because they are both aromatic and quite similar. Also, you might get away with a glycine being mutated to an alanine. Again, they're very similar.

 

But the amino acids outside of the active site are still crucial. If you imagine an enzyme being divided (for simplicity's sake) into two halves: a catalytic core, and the other half. The "other half" might not look very interesting, but if there's a substitution of a isoleucine to a proline, it will likely destroy any Beta-sheet structure, resulting in an altered overall shape. The catalytic core (active site) will be truncated (through loss of molecular architecture) and likely rendered nonfunctional.

 

As an example, there was a very interesting paper in Science from 1994, which investigated how acetylcholine is broken down by acetylcholinase in the brain. As it turns out, a couple of histidine residues and a few acidic residues in the catalytic site were responsible. But it didn't explain how the acetylcholine actually got in the active site - it was buried in the middle of the enzyme, with no apparent 'gap' for the AcChl to penetrate.

 

They eventually found a "back door" completely separate from the inner active site. It was on the "back" of the enzyme, and consisted of a cluster of charged groups which basically attracted the acetylcholine into the centre of the molecule. It was remarkable and novel, and even more progress has been made on this enzyme.

 

So what am I trying to say with this disjointed, nonsensical rant!? Well, I'd encourage you to consider the enzyme as a whole and not just the active site. Consider how all the amino acids will contribute not just to the chemistry of the active site, but also to the architecture of the protein itself. Residues outside of the active site will have surprising roles aiding the function of the enzyme, such as a 20-25 stretch motif of glycines and alanines being used to bind DNA in a nuclease.

 

So in a way you have nearly all the amino acids contributing to the function of the enzyme: catalysis. After understanding the overall portrait of the protein's chemistry, it's then a good idea to consider the active site.

 

As a good example of a well-studied active site, you would to well to look up the protein subtilisin. It's been commercially exploited and so lots of money has been invested into analysing its chemistry. In a nut-shell, you have a catalytic triad in the active site. It's a peptidase. I would draw a diagram, but I don't know how. Sooo..

 

To quote Wikipedia(!), "The charge-relay network functions as follows: The carboxylate side chain of Asp-32 hydrogen bonds to a nitrogen-bonded proton on His-64's imidazole ring. This is possible because Asp is negatively charged at physiological pH. The other nitrogen on His-64 hydrogen bonds to the O-H proton of Ser-221. This last interaction results in charge-separation of O-H, with the oxygen atom being more nucleophilic. This allows the oxygen atom of Ser-221 to attack incoming substrates (ie. peptide bonds), assisted by a neighboring carboxyamide side chain of Asn-155."

 

I'm sure that it'll be easy to find academic journals more credible than Wikipedia. But that's still a good summary. For further details, there are some great Nature and Science papers on this catalytic chemistry. It's taught as a classic example in a great deal of university biochemistry courses.

 

Hope this helps!

[Fortissimo]

ok thanks pple