Activation mechanism of meprins, members of the astacin metalloendopeptidase family

Meprins are mammalian zinc metalloendopeptidases with protease domains structurally related to astacin, the prototype of the ''astacin family'' of metalloproteases. Mature, active astacins are produced by proteolytic removal of an activation peptide to generate a new NH2-terminal residue. Structural studies indicate that the NH2-terminal ammonium group inserts into a water-filled cavity adjacent to the active site to form a salt bridge with a Glu residue that is conserved in all astacins. A similar interaction is known to play a crucial role in the activation of trypsin, resulting in the hypothesis that this salt bridge is required for the activation of astacin-like proteases. In this study, we have used the mouse meprin alpha subunit as a model to test this hypothesis of zymogen activation of the astacins. Mutants were generated to vary the NH2-terminal residue of the mature meprin alpha subunit (Asn(78)) and its putative salt bridge partner (Glu(178)). In addition, mutants-creating NH2-terminal extensions and truncations were expressed in human embryonic kidney 293 cells. The recombinant proteins were activated by limited protease digestion and assayed for enzymatic activity and thermal stability. Point mutations of Asn(78) resulted in enzymes with activity comparable to the wild-type enzyme, indicating that the structure of this side chain is not essential for activity. NH2-terminal extension mutants of meprin alpha retained partial activity, with greater decreases against peptide relative to protein substrates. A mutant with a deletion of Asn(78) to disrupt salt bridge formation with Glu(178) had full activity, indicating that the putative salt bridge with Glu(178) is not essential for enzyme activity. However, all changes in meprin alpha subunit NH2-terminal structure were found to decrease the thermal stability of the enzyme. These observations and additional data indicate that the zymogen activation mechanism of meprin and other astacins differs from that of the trypsin family of enzymes, and has some features in common with matrixins. It is proposed that prosequence removal of astacins allows the formation of hydrogen bonds involving the two NH2-terminal residues that are critical for enzyme structure.