title: Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber.
abstract: The activity of proteinase K (EC 3.4.21.14), a subtilisin-related serine proteinase, was assayed with azoalbumin that showed non-expected behavior in substrate saturation curve because of interaction between albumin molecules. Succinyl-(Ala)n-p-nitroanilide with n = 2 and 3, yielded specific activities of 3.5, 13 units/mg protein, respectively, reflecting a chain length dependence. The influence of peptide chain length on binding to proteinase K was also observed using mono- and dipeptide chloromethyl ketone inhibitors. They showed a maximum inhibition. They showed a maximum inhibition of proteinase K in solution of only about 50% even at a more than 20-fold molar excess. With the above substrates, the Vmax is not affected in presence of 10, 20 and 30% methanol, but the Km differs remarkably, suggesting competitive inhibition. The activity of proteinase K shows a maximum at 37 degrees C, and a temperature profile with more than 80% maximum activity in the range 20-60 degrees C. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 mg/ml) in aqueous solution. It does not occur at higher proteinase K concentrations at or above 1.0 mg/ml, consistent with crystallographic studies.
authors.affiliation: Institut für Kristallographie, Freie Universität Berlin, F.R.G.
authors.forename: J
authors.initials: J
authors.lastname: Bajorath
authors.forename: W
authors.initials: W
authors.lastname: Saenger
authors.forename: G P
authors.initials: GP
authors.lastname: Pal
mesh_terms: D001665:Binding Sites; D019286:Endopeptidase K; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013381:Subtilisins; D013816:Thermodynamics
chemical_list: D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D013381:Subtilisins; D019286:Endopeptidase K