Modulation of α-chymotrypsin specificity induced by pyridoxal

Summary Chymotrypsin catalyses the hydrolysis of the D-isomers of aromatic amino acids and of glycine methyl esters provided that pyridoxal is present. The corresponding L-isomers still behave as substrates for the enzyme even if pyridoxal decreases the rate of their hydrolysis. This change of enzyme stereospecificity should be taken into account in biotechnological processes.     

Self-association of α-chymotrypsin: Effect of amino acids

The concentration-dependent self-association of α-chymotrypsin is known to be influenced by various factors including the presence of small molecules and autolysis products. In this connection the effect of various amino acids on the self-association of α-chymotrypsin has been studied, as a point of interest, by measuring the sedimentation coefficient of α-chymotrypsin. The influence of an amino acid is seen to be governed by the nature of its side chain. Some amino acids do not affect the self-association of α-chymotrypsin at all while some affect it moderately and some others considerably. Functional groups such as the - OH group of Ser or the phenolic ring of Tyr do not seem to influence self-association behaviour. Based on these effects, amino acids could be categorized into 3 groups. Activity studies in the presence of amino acids indicate that the site of self-association and the active-site are probably mutually exclusive.     

Stability of immobilized α-chymotrypsin

The thermal of free and immobilized α-chymotrypsin was investigated experimentally and theoretically. The inactivation process of free α-chymotrypsin was analyzed with a kinetic model which included a first- order reaction process and autolysis. The effects of ionic strength, Ca²⁺ concentration, and temperature are discussed here in terms of the estimated kinetic parameters included in this model. The inactivation process of α-chymotrypsin immobilized onto various supports by several methods was investigated. The Contribution of thermal denaturation and autolysis to the inactivation depended upon the method of immobilization. To interpret quantitatively the non-first-order thermal denaturation process of the immobilized enzyme, a model in which the heterogeneity of the immobilized enzyme was taken into account is proposed.     

Stereoselectivity and reactivity of α-chymotrypsin modified at methionine-192

Native α-chymotrypsin (N-Chtr) and two modified forms, methionine-192 sulfoxide (O-Chtr) and methionine-192-S-(N-2-carboxyisopropyl)carbamylmethyl (Al-Chtr) α-chy-motrypsin have been compared in hydrolysis of methyl β-phenylpropionate and its d-and l-α-substituted derivatives, in which substituents are Cl, OH, OCOCH₃, NHCHO, and NHCOCH₃. In general, both modifications lower the reactivity of the enzymes in terms of k_cat and K_m(app); the decrease is greater toward l than toward d-enantiomers, and stereoselectivity is decreased. It is proposed that the modifications change the methionine-192 side chain from hydrophobic to hydrophilic, bring it into solution, and enlarge the Met-192-Ser-214 passage in which α-substituents fit. This leads to weaker binding, allows more freedom of motion of substrates and decreases reactivity, while allowing easier access of d-α-substituents. The large modifying substituent of Al-Chtr counters this effect by steric interaction with the large d-α-acetoxy and d-α-acylamido substituents, while the small polar modification in O-Chtr favors hydrolysis of these d-substrates.