Orlowski, Marian; Cardozo, Christopher; Hidalgo, Maria Carmen; Michaud, Charlene

Biochemistry (1991), 30(24), 5999-6005 CODEN: BICHAW; ISSN: 0006-2960. English.

The finding that the activity of the multicatalytic proteinase complex (MPC) is greatly activated by low concns. of SDS and fatty acids led to the proposal that the proteolytic activity of the complex is latent and that activation is needed for expression of full activity. Kinetic examination of the nature of the latency with carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, a substrate cleaved by the peptidylglutamyl-peptide-hydrolyzing activity (PGPH activity) of the complex, showed that plots of velocity vs. substrate concentration yield sigmoidal curves, implying that plots of velocity vs. substrate concentration yield sigmoidal curves, implying the presence of .gtoreq.2 substrate-binding sites and the presence of cooperative interactions between the sites. Hill plots of log [v/(Vmax - v)] vs. log log [S] gave slopes with a Hill coefficient of 2.2-2.4, suggesting that >2 subunits are expressing the PGPH activity. At saturating substrate concns., SDS and lauric acid exposed a masked component of PGPH activity that was about equal in magnitude to the overt activity measured in the absence of these detergents, showing that under the latter conditions only about half of the enzyme activity is expressed. Activation by SDS and lauric acid was greater at low than at high substrate concns. and was associated with a shift of the substrate concentration at half-Vmax (apparent Km) toward lower values. The decrease in the apparent Km in the presence of SDS (but not in the presence of lauric acid) was associated with a decrease in cooperativity. The presence of .gtoreq.2 distinct PGPH activity components with different reactivities was also indicated by the finding of 2 distinct inactivation rate consts. in reactions with 3,4-dichloroisocoumarin, an irreversible inhibitor of the enzyme. About 80% of the overt activity was inactivated with a rapid 2nd-order rate constant, whereas the remainder was inactivated at a slower rate that was equal to the rate of inactivation of the masked activity exposed by SDS. The PGPH activity of the MPC was strongly inhibited by low concns. (10-6-10-7M) of proteins present in pituitary homogenates and also by .beta.-casein, lysozyme, and bovine serum albumin. The binding of these proteins to the enzyme caused a concentration-dependent shift of the S-shaped substrate saturation curves with an increase in apparent Km without change in Vmax, a finding that could indicate neg. allosteric effects or competitive inhibition. It was concluded that cooperative interactions between >2 subunits of the complex determine the expression of the PGPH activity of the MPC and that full expression of this activity results from conformational changes induced by SDS or lauric acid binding at a site or sites (allosteric sites) different from the substrate binding site. These phenomena are in a major way responsible for the apparent latency of the MPC.