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 famil y'' of metalloproteases. Mature, active astacins are produced by prote olytic removal of an activation peptide to generate a new NH2-terminal residue. Structural studies indicate that the NH2-terminal ammonium g roup 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 astaci ns. A similar interaction is known to play a crucial role in the activ ation 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 h ypothesis of zymogen activation of the astacins. Mutants were generate d 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 expres sed in human embryonic kidney 293 cells. The recombinant proteins were activated by limited protease digestion and assayed for enzymatic act ivity and thermal stability. Point mutations of Asn(78) resulted in en zymes with activity comparable to the wild-type enzyme, indicating tha t 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 bri dge with Glu(178) is not essential for enzyme activity. However, all c hanges in meprin alpha subunit NH2-terminal structure were found to de crease the thermal stability of the enzyme. These observations and add itional 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 p rosequence removal of astacins allows the formation of hydrogen bonds involving the two NH2-terminal residues that are critical for enzyme s tructure.