1,2-Epoxy-3- propane sites

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

Cyclization. Halohydrin. The elimination reaction of ethylene halohydrins on solid catalysts gives ethylene oxide or other products depending on the nature of the catalyst. Acetaldehyde was obtained using a solid acid catalyst supported on silica gel by halide-ion abstraction, ethylene from the less polar sites on metal surfaces by halogen-atom abstraction, and ethylene oxide from the basic sites by proton abstraction. Among some of the more unusual epoxides which have been formed by base treatment of the corresponding halohydrin are the polyfluorinated epoxides (89) and the C-labelled l-halogeno-2,3-epoxy[3- K )]propane (90) (X = Cl, Br, or I). 

Irreversible /3-Agonists. Bromoacetyl compounds such as A-bromo-acetylnorepinephrine have the same effect as bromoacetic acid (unpublished). The very low affinity of the compound toward the receptor demands the use of high concentrations of these ligands, thereby resulting in nonspecific alkylation of the protein membrane. l,2-Epoxy-3-norepine-phrine-propane (see synthetic procedure A) retains its properties as a full agonist but fails to form a covalent bond at or near the receptor site. 

The fractions listed in the second column of Table II (P-II-A-l-b and P-II-B-l-b) are composed of Asp, Thr, Ser, Gly, Val], Ile and Phe]. This fragment appears to correspond to the following sequence which includes the active site residue Asp-32, which was modified by 1,2-epoxy-3-(p-nitrophenyl)propane (26) ... 

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. 

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