Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis.

The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.