The three-dimensional structure of procarboxypeptidase A (PCPA) from porcine pancreas has been determined at 2 Å resolution and refined to a crystallographic R-factor of 0·198, with a root-mean-square deviation from ideal values for bond lengths of 0·015 Å and for angles of 2·1 °. It is compared with procarboxypeptidase B (PCPB) from the same tissue. The 94/95 residue activation segments of PCPA/PCPB have equivalent folds: an N-terminal globular region with an open sandwich antiparallel α/antiparallel β topology, followed by an extended α-helical segment, the connection to the enzyme. Alignment of the secondary structures of the activation segments of PCPA and PCPB (residues A1 to A99) indicates a two residue insertion between residues A34 and A35 and a C-terminal helix that is two turns longer in PCPA compared to PCPB. A deletion is observed between residues A43 to A45, the region containing the short 310 helix that covers the active site in PCPB. The globular region (A4 to A80) shields the preformed active center of carboxypeptidase A (CPA), but none of the residues involved in catalysis makes direct contacts with the activation segment. In contrast, subsites S2, S3 and S4 of the enzyme, involved in the binding of peptidic substrates, are blocked by specific contacts with residues AspA36, TrpA38, ArgA47, AspA53 and GluA86 of the activation segment. It has been described that several residues of CPA exhibit different conformations in the free enzyme compared to when substrate is bound: Arg127, Arg145, Glu270 and Tyr248. In PCPA all of these residues are found in the "active" conformation, as if substrate were actually bound. The presence of a ligand, tentatively interpreted as a free amino acid (Val) in the active center could explain this fact. The connecting region (A80 to A99), the target for proteolytic activation, establishes fewer contacts with the enzyme in PCPA than in PCPB. The activation segment of PCPA (A4 to A99) remains bound to the enzyme after the first trypsin cleavage between ArgA99-Alal probably due to the stability confered on it by the α-helix (α3) of the connecting segment. These and other structural features may explain the differences in intrinsic activity and different rates or proteolytic activation of each zymogen. © 1992.
|Journal||Journal of Molecular Biology|
|Publication status||Published - 5 Mar 1992|
- inhibition and activation mechanism
- procarboxypeptidase A
- three-dimensional structure