Specificity of a neutral Zn-dependent proteinase from Thermoactinomyces sacchari toward the oxidized insulin B chain.

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the alpha-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15-Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T sacchari is different from those of other metalloendopeptidases from microorganisms.

A novel thermostable neutral proteinase from Saccharomonospora canescens.

A novel thermostable neutral proteinase, called NPS, was purified to electrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5. The molecular mass was determined by SDS-polyacrylamide gel electrophoresis to be 35,000 Da. The enzyme exhibits a sharp pH optimum of proteolytic activity at pH 6.7. NPS was completely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine proteinase inhibitor PMSF had no effect. Atomic absorption measurements showed that the proteinase binds a single zinc and four calcium ions. The enzyme thermostability was characterized in the absence and presence of added calcium. Melting temperature, Tm = 77 degrees C and an activation energy, Ea, for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol-1 were calculated in the presence of 100 mM CaCl2. The Ea-value is considerably higher than those obtained for a number of proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed. 68% of the light absorbed by tyrosyl residues is transferred to tryptophyl side chains. No homology was found after comparison of the NPS N-terminal sequence, including the first 26 residues, with those of other neutral proteinases from microorganisms. In contrast to the well-known bacterial neutral proteinase thermolysin and related enzymes from microorganisms, NPS possesses arylamidase and esterase activities. Further crystallographic studies will reveal the structural reasons for this specificity. Epoxy and epithio pyranosides are inhibitors of the proteinase arylamidase activity.