[Possible mechanisms of mechanical enzyme inactivation]. / O vozmozhnykh mekhanizmakh mekhanicheskoi inaktivatsii fermentov.

The changes of trypsin structures during grinding have been studied. Destruction of covalent bonds inside the polypeptide chain and of disulfide bonds was discovered by ESR method. Grinding at room temperature is accompanied by hydrolysis of peptide bonds which is demonstrated by the formation of a great number of new N-terminal amino acids, determined by reaction with dansyl chloride. A change in trypsin tertiary structure resulting from grinding was shown by circular dichroism method. The analysis of the results obtained allows to conclude that the main cause of inactivation in the course of enzyme grinding are the conformational changes due to rupture and redistribution of weak bonds.

[Enzyme inactivation after exposure to mechanical factors]. / Inaktivatsiia fermentov pri mekhanicheskikh vozdeistviiakh.

Kinetics of decreasing proteolytic activity of subtilisin and trypsin during grinding of powders was studied. The enzymes were ground in a ball laboratory vibromill for 1-80 min within the temperature range 80-300 K in the air or helium. Proteolytic activity of the enzymes measured by the splitting of low molecular p-nitroanilide substrates decreases with the increase of the treatment time according to the equation of the first order. By means of titration of trypsin active centres a decrease of their number after grinding was shown. Kinetic parameters in Michaelis-Menten equation were determined for trypsin enzymic reaction. It was found that the value kappa kat decreased with an increase of the time of mechanical treatment.

[Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates]. / O substratnoi spetsifichnosti vnutrikletochnoi sepinovoi proteinazy v Bacillus subtlis. Proteoliz v-tsepi insulina i ribonukleazy A i kinetika gidroliza p-nitoranilidnykh substratov.

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.