alpha-chymotrypsin and phenylethane-boronic-acid

A 1.8-A resolution x-ray crystallographic restrained least squares refinement has been carried out on the phenylethane boronic acid (PEBA) complex of alpha-chymotrypsin dimer (alpha-CHT), and it has been compared to the 1.67-A resolution structure of the native enzyme. PEBA has a high binding affinity for alpha-CHT, and the boronate forms a tetrahedral complex with Ser-195 OG of one molecule of the dimer; the boronate in the other molecule is severely disordered and does not form a tetrahedral complex. The former could be a model of the transition state of catalysis. The complex of PEBA X alpha-CHT displays significant nonequivalence in conformation of side chains between the independent molecules comparable to the native enzyme, but, like the latter, shows a high degree of fidelity in the folding of the main chain. The orientation of the phenyl ring, CA and CB of PEBA, in the specificity sites of the two molecules is similar, suggesting that recognition is fairly insensitive to small departures from local symmetry; the same does not apply to the boronate functionalities suggesting that greater precision is required for catalysis. The folding of the molecule remains the same upon PEBA binding, but some of the side chains respond nonequivalently. The latter is a consequence of the inherent nonequivalence of the native dimer and the asymmetrical nature of the PEBA binding.

Topics: Binding Sites; Boronic Acids; Chymotrypsin; Macromolecular Substances; Models, Molecular; Molecular Conformation; Protein Binding; Protein Conformation; X-Ray Diffraction

Several compounds have been tested for their ability to inhibit bovine pancreatic alpha-chymotrypsin (Ki) and their ability to inhibit cell replication (IC50). There is good agreement over three orders of magnitude between the Ki and the IC50 values of these compounds. The data support the hypothesis that a cellular, chymotrypsin-like activity is necessary for cell replication.

Topics: Animals; Boronic Acids; Cattle; Cell Division; Cell Line; Chymotrypsin; Kinetics; Mice; Pancreas; Protease Inhibitors; Structure-Activity Relationship

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