1.You are studying the world’s smallest enzyme which has the amino acid sequence

Question

1.You are studying the world’s smallest enzyme which has the amino acid sequence of:

Met Ala Ser Met Tyr Ala Glu

You know that activity of this enzyme requires phosphorylation. You wish to design an inactive mutant form of this enzyme that can never be phosphorylated. You will use site-directed mutagenesis to alter the DNA sequence of this protein, resulting in a mutant protein.

Additional information: The most common resides to be phosphorylates are serine, threonine and tyrosine. In theory, these can be replaced with any other amino acid except for glutamic and aspartic acid, the shape and charge of a phosphorylated serine and aspartic acid are similar. Substitution with one of these amino acids would create a protein that looked like it was always phosphorylated (phosphomimic). Alanine is one of the most common substitutions, but others are possible and sometimes preferable to preserve other characteristics of the amino acid.

1. Write out the resulting amino acid sequence corresponding to your designed mutant protein here:

Explanation / Answer

In the evolution of proteins more specifically between the division of prokaryotes and Eukaryotes it has been studied that aspartic acid and glutamic acid found on the surface; for example, in a salt bridge that stabilize a fold that is necessary for proper function. A mutation causes negatively charged glutamate to be replaced by a neutral serine. This destabilizes the salt bridge, rendering the protein inactive. Now this is a evolutionary mechanism that works for temporal and spatial regulation of the necessary protein in the cell, the kinases are regulatory enzymes that are able to phosphorylate the serine/The/Tyr, the negatively charged phosphate could reestablish the salt bridge, stabilize the fold, and reactivate the protein. Due to phosphates charged groups the pSer and pThr provide the necessary negative charge just like Glu and Asp.

In site directed mutagenesis the aim is to study the importance of a single aminoacid by mutating it or replacing it, now there are certain rules for the replacement of a aminoacid residue in concern; it should be always replaced by smaller side chain residue because larger side chains can cause steric hindrances and destabilize the structure of the protein. Various bioinformatics methods have made this process easy and given the possible replacement aminoacid tables.
Accordingly if we want to recreate a aminoacid sequence which cannot be activated by phosphorylation, we first identify the aminoacids that are site of phosphorylation and replace it with the aminoacid that cannot be phosphorylated by any kinases.
So, in our sequence we have 2 phosphorylatable sites that is Serine and Tyrosine.
Serine can be replaced with Ala, Asn and Gly.( Asn has big side chain so Ala and Gly are better candidates)
Tyrosine can be replaced with Phe, His and Trp.(Phe is more of a suitable candidate because it has smaller size that Tyrosine and lacks the OH group)
So the proposed sequence will be:

Met-Ala-Gly-Met-Phe-Ala-Glu

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