ACTIVATION AND CHARACTERIZATION OF PROCARBOXYPEPTIDASE-B FROM HUMAN PLASMA

Recently we reported the isolation and cloning of a novel plasma proca rboxypeptidase B that binds plasminogen [Eaten, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., & Drayna, D. (1991) J. Blob. Chem. 266, 21833 -21838]. This plasma procarboxypeptidase is structurally similar to ti ssue procarboxypeptidases, and initial substrate studies showed that t his plasma protein behaves like a basic carboxypeptidase and is now kn own as human plasma procarboxypeptidase B (pro-pCPB). However, unlike the tissue procarboxypeptidases, pro-pCPB is extremely unstable to try psin activation. Trypsin cleaves pro-pCPB at two sites: Arg-92 and Arg -330. Cleavage at Arg-99 releases the activation peptide and generates an active enzyme. However, cleavage at Arg-330 inactivates pCPB. This renders the characterization of pCPB difficult. We have found that 6- amino-n-hexanoic acid (epsilon ACA), a compeptitive inhibitor of basic carboxypeptidases, selectively limits trypsin cleavage of pro-pCPB. I n the presence of epsilon ACA, trypsin cleavage at Arg-330 is signific antly limited while the cleavage at Arg-92 is unaffected. Using this a pproach, active pCPB can now be obtained. Kinetic characterization sho ws that pCPB behaves like other known basic carboxypeptidases. pCPB is more specific for substrates with C-terminal arginine than those with C-terminal lysine for all the natural and synthetic peptides tested. It also hydrolyzes the synthetic ester substrate more efficiently than the synthetic peptide substrate, especially at high pH. The active si te Zn2+ can be replaced with other metals with change in substrate spe cificity. Binding studies using either Lys-plasminogen or Glu-plasmino gen with pro-pCPB or pCPB show that pro-pCPB has a 10-fold higher affi nity to both forms of plasminogen than pCPB. This suggests that the gl ycosylated activation peptide mediates the high-affinity binding of pr o-pCPB to plasminogen. Ligand Mot binding studies show that the bindin g site for pro-pCPB to plasminogen is inhibited by alpha(2)-antiplasmi n, suggesting a similar site of interaction.