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Unformatted text preview: of the specificity pocket by
removing the methyl group of Thr. This makes the pocket better fit the larger side chain
Leu relative to Val. The changes in KM may reflect better binding of Leu and worse
binding of Val. 9. Refer to the active site for thermolysin and the general mechanism for the
metalloproteases covered in the lecture notes. Describe how this enzyme utilizes the
following strategies to accelerate hydrolysis of the substrate: metal ion catalysis, acidbase catalysis, covalent catalysis and/or substrate proximity and orientation. Indicate
which, if any, of these are not used by thermolysin.
Metal ion catalysis: The enzyme uses a Zn cofactor to bind and activate a water molecule
which then acts as a nucleophile.
Covalent catalysis: Not present in this mechanism. The enzyme does not form covalent
bonds to the substrate.
Acid-base catalysis: Glu 143 deprotonates the water molecule to generate a hydroxide
nucleophile, which then attacks substrate
Proximity and orientation: the enzyme binds two substrates, water and the peptide. The
configuration of the active site binds them next to each other and in the right orientation
to react productively....
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- Spring '08