title: Enzymic and physicochemical properties of Streptomyces griseus trypsin.
abstract: Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.
mesh_terms: D000107:Acetylation; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004355:Drug Stability; D004950:Esterases / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013305:Streptomyces griseus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D014361:Trypsin Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D006639:Histidine; D004950:Esterases; D014357:Trypsin