Active-site-directed reagents other studies

On the other hand, there are good reasons to believe that the basic modes of inhibition of the tetrapeptide and of pepstatin are the same as that proposed above for the smaller statyl compounds (Fig.3). The "competitiveness and "transition state" hypotheses for pepstatin are both supported by x-ray crystallographic results (18 and Chapter 3 in this book) of R. chinensis in which the reactive site of an active center-directed reagent, l,2-epoxy-3-(p-nitrophenoxy)propane, overlaps with the pepstatin site. Further support comes from the studies of Rich al. (19). Dideoxypepstatin, which they synthesized, was shown to be a competitive inhibitor of pepsin, with = 2.1 x 10 M. This represents about a 2,000-fold increase in value caused by removal of the oxygen atoms from 3-position of statyl residues of pepstatin. 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

A structure-function study of a proton pathway in the y-class carbonic anhydrase from Methanosarcina thermophila was conducted in the work of Tripp and Ferry (2000). Four enzyme glutamate residues were characterized by site-directed mutagenesis. It was shown that Glu 84 and an active site residue, Glu 89, are important for CO2 hydration activity, while external loop residues, Glu 88 and Glu 89 are less important. Glu 84 can be substituted for other ionizable residues with similar pKa values and, therefore, participates in the enzyme catalysis not as a chemical reagent but as a proton shuttle. 

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