Comparative study on activation mechanism of carboxypeptidase A1, A2 and B: First insights from steered molecular dynamics simulations

Different forms of procarboxypeptidases (PCPs) zymogens are observed experimentally to show different rates and modes of activation: PCPA1 shows a slow, biphasic, activation pathway compared to PCPA2 and PCPB which have a faster, monotonic activation behavior. Detailed mechanisms involved in activating these zymogen forms to the active enzymes are not well understood yet. In this work, three PCP zymogens (subtypes A1, A2 and B) were in silico converted into the primary cleavage state of zymogens using available X-ray structures. Based on these cleaved forms of zymogen, we are able to investigate their spontaneous dissociation process of the prosegment from its associated enzyme domain using steered molecular dynamics simulation. The simulations revealed the highest rupture force in PCPB followed by PCPA2 and PCPA1. We also found that the cleavage substantially destabilizes most of the hydrogen bonds at the prosegment–enzyme interface in each zymogen structure. The mechanisms of the prosegment unbinding seem to be similar in both PCPA1 and PCPB but different in PCPA2: PCPA1 and PCPB show first rupture in the connecting segment followed by the globular domain, while PCPA2 conversely shows first rupture in the globular domain and then in the connecting segment. Our simulations have included the dynamic and long range conformational effects taking place after the first proteolytic cleavage in PCPs, providing first insights into the activation of carboxypeptidase A1, A2 and B.

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► Three procarboxypeptidases A1, A2 and B in a primary cleaved state were modeled.
► Steered MD simulation of the models was done to pull a prosegment out of the enzyme.
► The force profile and the rupture interaction for each simulation were analyzed.
► The prosegment unbinding mechanism is similar in PCPA1 and PCPB but different in PCPA2.