alpha-chymotrypsin and indole

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.

Topics: Animals; Catalysis; Cattle; Chymotrypsin; Electron Spin Resonance Spectroscopy; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized; Indoles; Membranes, Artificial; Models, Chemical; Pancreas; Sensitivity and Specificity; Sepharose; Structure-Activity Relationship

Topics: 1-Propanol; Binding Sites; Chymotrypsin; Electron Spin Resonance Spectroscopy; Enzymes, Immobilized; Hymecromone; Indoles; Kinetics; Spin Labels

The heat of formation of the chymotrypsin-phenylethane boronic acid complex has been observed calorimetrically from pH 4 to 8 at 25 degrees C and is found to be pH-dependent, changing from near -6 kcal/mol at pH 4 to -13 kcal/mol at pH 8. The heat of formation of the chymotrypsin-indole complex is a nearly constant -6 kcal/mol over most of the same pH range. alpha-Chymotrypsin has been purified by pH gradient elution from an immobilized lima bean inhibitor column. Solutions of the enzyme up to 400 microM, prepared in this manner, have a zero heat of dilution from pH 5 to 8 in 0.1 M KCl, with or without added 0.05 M Tris, N-(tris[hydroxy-methyl]methyl-2-amino) ethanesulfonic acid, 4-morpholineethanesulfonic acid, or acetate buffers. Binding of phenylethane boronic acid causes a pH-dependent decrease in proton binding to chymotrypsin; the decrease in proton binding evoked by formation of the indole complex is much less, with a much smaller pH dependence. The calorimetric and proton-binding results are applied to a model for boronic acid binding (Hanai, K. (1976) J. Biochem. (Tokyo) 79, 107-116). We conclude that the thermodynamics of formation of the trigonal boronic acid complex are quite similar to those for the formation of the noncovalent complex formed by indole and related ligands. The trigonal-tetrahedral tautomerism in the boronic acid-chymotrypsin complex is characterized by thermodynamic changes similar to those accompanying the binding of virtual substrates to chymotrypsin.

Topics: Boronic Acids; Calorimetry; Chymotrypsin; Hydrogen-Ion Concentration; Indoles; Mathematics

Topics: Amides; Carboxylic Acids; Catalytic Domain; Chemical Phenomena; Chemistry; Chymotrypsin; Enzyme Inhibitors; Hydroxamic Acids; Indoles; Ions; Kinetics; Research

Topics: Biophysical Phenomena; Chymotrypsin; Indoles; Sensitivity and Specificity