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Unformatted text preview: BL/CH401 Lecture 17 Mutagenesis & Enzyme Catalysis via Transition State Part I. Introduction I want to introduce the concept of site-directed mutagenesis and how this method is used to make mutant proteins for studying the biochemistry to an enzyme or protein. You must first recall that the AA sequence of a protein is encoded in the DNA sequence. So to make a mutant of the protein, one must change the DNA sequence and then express the protein in its mutant form. Overall, the process is to alter the nucleotide sequence of the DNA in vitro (ie outside the cell and actually in the lab) and I will not describe how one does that. Then the DNA is put into a bacterial cell to express the protein. Of course, the protein must be purified from the bacterial extract before it can be studied. Usually when mutants are made, they are studied in comparison to the natural or wild-type protein, as it is called. In Fig. 1 below, are shown examples of a mutant protein made by changing a single nucleotide base in the sequence of the DNA. In some cases, a base change does not lead to a new AA in the sequence since the genetic code is redundant (see lecture 2 for more information on the genetic code). Examples of the effects of changing a nucleotide or base in a DNA sequence encoding a protein: Here a single base is changed (always G to T) Wild Type GGC TTC CCG GTG CGC GTC ATC ATC CCC GGC TGC ATG G F P V R V I I P G C M Gly Phe Pro Val Arg Val Ile Ile Pro Gly Cys Met * G to T GGC TTC CCT GTG CGC GTC ATC ATC CCC GGC TGC ATG in G F P V R V I I P G C M 3rd base Gly Phe Pro Val Arg Val Ile Ile Pro Gly Cys Met of *** a codon Same AA * G to T GGC TTC CCG GTG CTC GTC ATC ATC CCC GGC TGC ATG in G F P V L V I I P G C M 2nd base Gly Phe Pro Val Leu Val Ile Ile Pro Gly Cys Met *** New AA * G to T GGC TTC CCG GTG CGC GTC ATC ATC CCC TGC TGC ATG in G F P V R V I I P C C M 1st base Gly Phe Pro Val Arg Val Ile Ile Pro Cys Cys Met *** New AA Figure 1. Mutagenesis of a protein by changing a single base. This is a very general example and the mutations are made at random. When carrying out site- directed mutagenesis, a very specific site in the protein and an individual AA is selected for mutagenesis. That is what makes this method so important to understand...using the method the investigator can actually choose the AA he/she wants to change and then change it to any of the other 19 AA's encoded by the codons in the genetic code. This process is very controlled and leads then to a way to gain much greater insight into protein structure and function because the impact of different AA side chains at the same site in a protein/enzyme can be studied and the importance of an individual AA side chain can be understood in some detail. Ultimately, it can be expected that this type of experiment will allow the investigator to actually design new functionality into old enzymes and even design new enzymes.In this lecture, I want to apply the concept of site-directed mutagenesis to the problem of understanding how an enzyme works as a...
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