The hydroxyl of the serine at the active site of subtilisin or trypsin could be converted to a sulfhydryl, and the sulfhydryl at the active site of papain converted to a serine residue. Although, as shown in Fig. 6-11, it has become possible to chemically alter serine or cysteine residues in active sites, other serine or cysteine residues may also be modified, and the harsh conditions employed for such alterations may lead to secondary reactions of other residues. In an approach designed to radically alter the activity of the parent protein, some recent chemical modification work has been directed toward derivatizing, for example, the active-site cysteine in papain with flavin coenzymes. Such methods are designed to create new enzyme activities rather than provide insight into the functioning of the parent protein. Genetic engineering techniques have allowed a wide range of such interconversions to be accomplished via a process known as site-directed mutagenesis.
Many methods exist by which mutations can be introduced into a specific region of DNA sequence.
Also, techniques are available to introduce specific mutations into a site where a restriction endonuclease can act. In general, two methods are available to introduce mutations at specific sites. The first involves the use of the single-stranded bacteriophage Ml 3. From the gene sequence of the protein around the residue to be mutated a nucleotide sequence encompassing two or three amino acid residues either side of the altered codon is derived and the oligonucleotide chemically synthesized. Some approaches employ two primers, but the basic theory is the same.