1. Acid proteases. II. Fluorescence study of the interaction of Cladosporium acid protease with glycyl-DL-norleucine methyl ester in the presence of cupric ions
H Kanazawa J Biochem. 1978 Mar;83(3):665-9. doi: 10.1093/oxfordjournals.jbchem.a131958.
Glycyl-DL-norleucine methyl ester (GN), a diazoacetyl-DL-norleucine methyl ester (DAN) analog, in the presence of cupric ions was found to partially quench the protein fluorescence of acid protease from Cladosporium sp. No. 45-2, and cupric ions were also found to quench the fluorescence. These quenchings were pH-dependent. GN alone did not quench the fluorescence of the enzyme. The interaction between the enzyme and GN in the presence of cupric ions was studied statically at pH 5.4 in terms of fluorescence change. The dissociation constant, Kd, of the enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (0.08 mM) determined by fluorescence titration at 30 degrees C (Kd = 1.86 mM) was in good agreement with that obtained for GN from kinetics of inhibition of DAN-induced inactivation in the presence of a 20-fold molar excess of cupric ions at 30 degrees C (KA = 1.94 mM) (Kanazawa, H. (1977) J. Biochem. 81, 1739-1744). At various concentrations of cupric ions, no change of Kd was found. These results suggest that cupric ions are attracted to a negatively charged carboxyl group responsible for the formation of the enzyme-GN complex.
2. The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin
K Takahashi, W J Chang J Biochem. 1976 Sep;80(3):497-506. doi: 10.1093/oxfordjournals.jbchem.a131304.
Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.
