The interaction of inhibitors of proteolytic enzymes with 3-methylhistidine-57-chymotrypsin.

The inhibition of proteolytic enzymes by protein inhibitors is accompanied by the formation of unusually stable complexes. The recognition of a specific substrate-like amino acid on the inhibitor is believed to be the initial event of the inhibitory process. In addition to the interactions involved in the binding of a good substrate, a variety of other noncovalent interactions are known to stabilize the complex. The formation of stable complexes between several inactive derivatives of proteolytic enzymes and a variety of protein inhibitors suggests strongly that the formation of any species resembling catalytic intermediates is unnecessary for inhibition. We have examined the interaction between several avian ovomucoids and alpha-chymotrypsin in which histidine-57 has been converted to 3-methylhistidine-57. This derivative is easily prepared and can be isolated by affinity chromatography. Methylchymotrypsin retains unaltered its ability to bind specific substrates, but is essentially inactive. In spite of this loss of enzymatic activity, methylchymotrypsin forms strong complexes with several inhibitors. In addition, methylchymotrypsin which has been covalently linked to Sepharose is particularly useful for the isolation of protein inhibitors without the complications due to isolation of a mixture of partially cleaved forms of the inhibitor.     

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.