The Proteolytic Enzymes of the K-1 Strain of Streptomyces griseus Obtained from a Commercial Preparation (Pronase)

• Published in: The Journal of biological chemistry 1974-10, Vol.249 (19), p.6144-6148
• Main Authors: Russin, David J., Floyd, Benjamin F., Toomey, Thomas P., Brady, Al H., Awad, William M.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42232-1

Streptomyces griseus trypsin is more thermolabile than the two other components in pronase which are homologous with bovine chymotrypsin. It is completely inactivated after heating to 60° for 15 min. The heat stability of the enzyme is reduced in the presence of EDTA. Calcium was found to be the specific cation which stabilizes the enzyme at higher temperatures. This trypsin denatures irreversibly in 8 m urea (at 23°) in low calcium ion concentrations, but is stable and active in this denaturant if 0.5 m calcium ion is present. This latter property makes this enzyme a possibly useful agent in protein structural studies. Both the microbial and bovine trypsin are found to bind guanidinium ion substantially. Guanidinium ion competitively inhibits the activity of each enzyme against Nα-benzoyl-l-arginine-p-nitroanilide. Microbial trypsin has about a 3-fold greater affinity for guanidine and about a 20-fold lower Km for Nα-benzoyl-l-arginine-p-nitroanilide than does bovine trypsin. Binding of guanidine with either enzyme produces no apparent inhibition of activity against the poor nonspecific substrate, p-nitrophenyl acetate, when compared to inhibitorfree solutions. These findings suggest that guanidine associates with that part of the specificity site which binds the charged portion of basic substrate residues. The addition of 0.2 m guanidine HCl to an 8 m urea-10 mm CaCl2 solution completely inhibits the autolysis of the microbial trypsin but only slightly decreases the rate of autolysis of the bovine enzyme. In 8 m urea-10 mm CaCl2 and 1.0 m guanidine HCl, about 90% of the activity of the microbial enzyme is retained after 2 hours even in the presence of another S. griseus serine endopeptidase known to be active and stable in this mixed denaturant solution. Therefore, guanidine appears to stabilize microbial trypsin. In the presence of sodium ethylenediaminetetraacetate and denaturant mixture, the microbial enzyme rapidly loses activity. Measurements of circular dichroism were made at pH 8 and revealed that 0.45 m CaCl2 completely protects the microbial enzyme against rapid unfolding by 8 m urea, whereas this salt has little effect upon the rapid conformational transition of the bovine enzyme in this denaturant. It was found also that 0.2 m guanidine with a low calcium ion concentration can largely stabilize in 8 m urea the conformation of the microbial enzyme. A study was carried out to see if the guanidine complex of either trypsin could demonstrate a cha