Structural Biochemistry/Enzyme/Site-Directed Mutagenesis

=Site-Directed Mutagenesis=

Mutagenesis is a broad term that is defined as the alteration of the genetic material of an organism in a stable manner.

Site-directed mutagenesis is when the amino acid sequence of a given enzyme molecule or other protein may be altered by deliberately and precisely mutating the cloned gene encoding that molecule. It is a very useful technique that can be used in the study of protein function, the identification of enzymatic active sites, and the design of novel proteins. With this technique, it is possible to exchange a single amino acid in the sequence of a protein for another amino acid with different chemical properties. In this way, the function of the specific amino acid at this site can be examined. The basic protocol for this process was developed by Michael Smith, who was awarded the Nobel Prize in Chemistry in 1993 "for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies" (1)

In order to carry out site-directed mutagenesis, a DNA primer must be designed at the site of interest. The primer should contain the necessary nucleotide differences in order to affect the change in the protein sequence. For example, consider the case where a protein sequence reads Tyr-Leu-His-Val, corresponding to a genetic sequence of UACCUGCACGUC. If the experimentalist intends to mutate the histidine residue to a leucine residue, they might design a primer with the sequence UACCUGCUCGUC. This primer is then hybridized to the complementary single stranded DNA molecule and extended using a DNA polymerase. A mutated double stranded DNA molecule encoding the (mutated) protein is obtained in this way, and this DNA molecule is cloned into a host cell. Host cells are allowed to grow and mutants are selected. In this way, proteins that are generated by this DNA sequence will contain the desired mutation. A similar procedure can be carried out with the use of PCR (PCR site-directed mutagenesis)



An example of site-directed mutagenesis: catalytic triad
Site-Directed Mutagenesis is a method used to dissect the amount of catalytic power by each of the catalytic triad in a enzyme. This is done by converting each of the triad into a common amino acid and measure the catalytic power differences.

For example, in subtilisin, the catalytic triad that is studied by this method are aspartic acid 32, histidine 64, and serine 221. Each of the amino acids in the triad will be converted into alanine and thus, the ability of the mutant enzyme to cleave a substrate is examined. The result by this method shows that serine 221 into alanine reduces the catalytic power as much as histidine 64 into alanine. The value of the kcat for serine 221 and histidine 64 becomes one-millionth of its original value. As for aspartic acid 32, the catalytic power is reduced but not as much as for serine 221 and histidine 64. The kcat value is 0.005% of the original enzyme. Thus, the result of using the site-directed mutagenesis shows that the serine-histidine pair makes a nucleophile that has the capability to attack the carbonyl carbon atom in the peptide bond. Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are a number of methods for achieving this. The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found here. Even when using a kit it will be necessary to design primers that are suitable for the specific changes you want to make to your DNA. Most of the contents of the kit can be found in your favorite labs stocks so you may not need to buy the kit itself. If you have problems with this procedure, you can try 'Round-the-horn site-directed mutagenesis which uses PCR to amplify the desired mutant product.



Mutagenesis has been used in terms of DNA recombinant technique.