alpha-chymotrypsin and benzoylphenylalanine

alpha-Chymotrypsin, when immobilized in a collodion membrane, exhibits high activity and remarkable stability. When the immobilized proteinase is exposed to 15 mM ethyl N-acetyl-L-tyrosinate in dilute pH 8.5 buffer it generates a microenvironment which, indicator studies suggest, has an effective pH of approximately 4. The presence of this locally highly acidic region produces a marked increase in the rate of hydrolysis of BzPheal = Ala dissolved in the buffer solution (BzPheal = Ala is the acylhydrazide obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide). The observed rate is 10-times greater than in comparable control experiments incorporating a concentrated buffer solution, in which a pH-gradient does not form. The enhanced hydrolysis rate is quantitatively explained if it is attributed to the approximately 20 microliters of pH 4 solution within the membrane. Other experimental data are also consistent with this hypothesis.

Topics: Alanine; Buffers; Chemical Phenomena; Chemistry; Chymotrypsin; Collodion; Diffusion; Enzymes, Immobilized; Hydrazines; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Phenylalanine; Prodrugs; Solutions; Tyrosine

The reaction between peptide aldehydes and acylhydrazones affords derivatives that represent potential prodrugs for selective inhibition of lysosomal enzymes. BzPheal = Ala, obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide, has been most carefully studied. When BzPheal = Ala is introduced into ongoing reactions catalyzed by alpha-chymotrypsin or papain, the rate of these reactions diminishes more rapidly with time than do those of controls lacking BzPheal = Ala. Furthermore, the disparity between run and control is much greater at pH 5 than at pH 7. The extent of inhibition (defined as explained in the text) at pH 5 can exceed that at pH 7 by 25-40-fold. The data are quantitatively explained by a reaction scheme that recognizes three important properties of BzPheal = Ala: (1) It undergoes hydrolysis at pH 5-7 to regenerate N-benzoyl-L-phenylalaninal; (2) the aldehyde thus liberated is a far more potent inhibitor for serine or cysteine proteases than is BzPheal = Ala; and (3) the rate constant for hydrolysis of BzPheal = Ala at pH 5 greatly exceeds that at pH 7.

Topics: Aldehydes; Chemical Phenomena; Chemistry; Chymotrypsin; Hydrazones; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lysosomes; Papain; Phenylalanine; Prodrugs; Protease Inhibitors