Cysteine using site-directed mutagenesis

Hatfull, G. R, Sanderson, M. R., Freemont, P. S., Raccuia, P. R., Grindley, N. D. F. and Steitz, T. A. (1989). Preparation of heavy-atom derivatives using site-directed mutagenesis introduction of cysteine residues into yS resolvase. /. Mol. Biol. 208,661-667. 

Protein engineering has also been used to alter the co-factor preferences of XR and XDH. Inhibition studies of P. stipitis XR suggested that histidine and cysteine residues might be involved in co-factor binding [145]. Using site-directed mutagenesis, the three cysteine residues were individually changed into serine... 

Clark KM, Yu Y, Marshall NM, Sieracki NA, Nilges MJ, Blackburn NJ, van der Donk WA, Lu Y. Transforming a blue copper into a red copper protein engineering cysteine and homocysteine into the axial position of azurin using site-directed mutagenesis and expressed protein ligation. J Am Chem Soc 2010 132 10093-10101. 

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

Site-directed mutagenesis is one of the most powerful methods of studying mechanisms of enzyme-catalyzed reactions. Since this technique makes it possible to replace a specific amino acid residue of an enzyme by an arbitrary one, it is particularly useful to specify the amino acid residue(s) which is responsible for the activity [20 - 22]. In the case of AMDase, one of four cysteine residues was presumed to be involved in the catalytic site by the titration experiments. To determine which Cys is located at the active site, preparation of four mutant enzymes, in each of which one of the cysteines is replaced another amino acid, and kinetic studies on them, are expected to be most informative. Which amino acid should be introduced in place of cysteine To decide on the best candidate. 

Some examples of hydrolases are listed in Table 7.3 according to the industrial sector in which they are used. Most of these hydrolases operate in the hydrolytic mode, since only a few processes are currently known that make use of the synthetic power of hydrolases. Increasingly, however, these enzymes are being tailored by genetic tools such as random or site-directed mutagenesis and overexpression to lower the operational costs of the biocatalyst and to obtain enzymes of high purity, as well as to meet specific customer requirements such as stability, substrate specificity, and optimal pH and temperature of operation. A typical example is the protease subtilisin from Bacillus subtilus, which was made bleach-resistant by replacing one bleach-sensitive amino acid (cysteine) by the chemically inert alanine [12]. 

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