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lrob lrob
wrote...
Posts: 30
Rep: 2 0
12 years ago
Hey!

I've been reading this paper which describes the structure of an intracellular subtilisin protease from Bacillus clausii, which is a type of subtilisin-like serine protease (as opposed to a trypsin/chymotrypsin serine protease). Subtilisin-like serine proteases usually have a conserved catalytic triad of Asp-32, His-64 and Ser-221, however in this paper, the protein seems to have an active site of Asp-49, His-86 and Ser-250. Why is this?

In addition, for the purposes of stabilising this protein for study, the active serine was mutated to Ala-250, could this have anything to do with the location of the active site?

Any help would be greatly appreciated, Thanks!

Vévodová, J., Gamble, M., Kunze, G., Ariza, A., Dodson, E., Jones, D. D. and Wilson, K. S. 2010. Crystal Structure of an Intracellular Subtilisin Reveals Novel Structural Features Unique to this Subtilisin Family. Structure. 18(6), pp. 744-755.

http://www.sciencedirect.com/science/article/pii/S0969212610001401
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wrote...
12 years ago
Subtilisin-like serine proteases usually have a conserved catalytic triad of Asp-32, His-64 and Ser-221, however in this paper, the protein seems to have an active site of Asp-49, His-86 and Ser-250. Why is this?

What do you mean by "why is this"? It's normal for these amino acids to shift their positions in a protein.

In addition, for the purposes of stabilising this protein for study, the active serine was mutated to Ala-250, could this have anything to do with the location of the active site?

As far as I can remember, this is a highly active site, therefore, the amino acid must be replaced in order for the researchers to have control of that area.
Biology!
lrob Author
wrote...
12 years ago
Ok, so if active sites move around, then why is it said that subtilisin proteases have a conserved active site of Asp-32, His-64 and Ser-221? If movement of active site is a normal thing to happen, why not refer to the protein's active site being composed of Asparagine, Histidine and Serine without mentioning their residue number?

I understand why the Ser-250 was mutated to Ala-250, I just want to know whether the act of this point mutation somehow affected the location of the active site.

Thanks!
wrote...
12 years ago
Ok, so if active sites move around, then why is it said that subtilisin proteases have a conserved active site of Asp-32, His-64 and Ser-221?

I didn't mean it that way. The gene that codes for this active site could be different from one organism to the other. The sequence in bacteria A could be slightly different than the sequence in bacteria B. Yes, this active site is conserved in many organisms in the sense that it works the same fashion, where ultimately a triad is formed. That's my take on it anyway and it's how I've understood it for the longest time.
Biology!
lrob Author
wrote...
12 years ago
Ok thanks, that's a great help and makes sense, it does seem odd though that it's written that the subtilisins have a conserved active site of Asp-32, His-64 and Ser-221 when not all subtilisins do. Thanks!
wrote...
12 years ago
I understand why the Ser-250 was mutated to Ala-250, I just want to know whether the act of this point mutation somehow affected the location of the active site.

Both these amino acids look very similar, but it's enough to prevent a reaction from taking place.

See: alanine
serine
Biology!
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