ses13_ln

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MIT OpenCourseWare http://ocw.mit.edu 7.344 Directed Evolution: Engineering Biocatalysts Spring 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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Session 13 Lecture Notes 1. PTE – we have seen this enzyme before – is phosphotriesterase. Its capabilities to hydrolyze organophosphates like sarin, VX and soman make it of particular interest. The enzyme uses a binuclear zinc metal center to activate a water molecule. This water molecule acts as a nucleophile toward the substrate and cleaves the e- group leaving a phosphate (Not harmful) behind. Why are these organophosphate materials not harmful to PTE? It is water that does the chemistry, not an enzyme side chain so the substrates do not act as suicide inhibitors. The zinc residues are held in place by two histidines each and two aspartates that bridge the zinc ions. See Figure 1 panel A. 2. The authors use structure-guided cassette mutagenesis to mutate the residues above the binding pocket (H254, H257) as they are known to contribute to substrate selectivity. They get a library of 400 variants and screen 1200 colonies using HTS for desired catalytic activity. The authors choose this method because much is already know about the enzyme mechanism. The then use their best guys and make a new site-saturation library at another position selected using structural data – iterative cassette mutagenesis. 3.
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ses13_ln - MIT OpenCourseWare http:/ocw.mit.edu 7.344...

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