Abstract
1. Bovine rennin [EC 3. 4. 4. 3] was rapidly inactivated in 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. It was also inactivated by reaction with l, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with concomitant incorporation of two equivalents of the reagent into the enzyme. The DAN-inactivated enzyme and the EPNP-inactivated enzyme also reacted with EPNP and DAN, respectively, in the same way as native rennin. The reagent moieties incorporated into the enzyme were labilized by treatment with aqueous hydroxylamine. These results indicate the implication of two distinct carboxyl groups in these reactions, and hence in the active site of rennin.
2. Pepstatin, a specific inhibitor of pepsin [EC 3. 4. 4. 1], inhibited rennin over a wide pH range (pH 2.0-6.0). It also significantly inhibited the reactions with rennin of diazoacetyl-DL-norleucine methyl ester and 1, 2-epoxy-3-(p-nitrophenoxy) propane. These results indicate that pepstatin binds to at least part of the active site of rennin. p-Bromophenacyl bromide, a specific inactivator of pepsin, failed to inactivate rennin.
