Chymotrypsin water binding

Blevins and Tulinsky (1985) made the suggestion, based on a 1.67 A resolution structure for qe-chymotrypsin, that several molecules of the specificity-site water are not displaced on substrate binding and may serve to position the substrate. 

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

Experiments performed in a very similar way also showed dynamic disorder for the enzyme a-chymotrypsin [16], a-Chymotrypsin is an endopeptidase acting on water-soluble polypeptides. The substrate (suc-AAPF)2-rhodamine 110 was designed to interact optimally with the binding site of the enzyme. It consists of a rhodamine 110 core that is derivatized with a succiny-lated AlaAlaProPhe peptide sequence, known to bind very specifically at the enzyme s active site (Fig. 25.2a). To further avoid potential artifacts, the enzyme was immobilized by entrapment in an agarose matrix, which restricts enzyme diffusion while still allowing free rotation and conformational dynamics of the enzyme as well as the diffusion of the substrate. 

Finally, a molecule of water participates in the chemistry of catalysis. The HO" group attacks the covalently bound part of the substrate (dietary protein), resulting in its discharge fmm the chymotrypsin molecule (Figure 2.WF). The H of water then binds to Ser 195, regenerating the serine group to its iriitial non-ionized state. 

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