An unusual active site identified in a family of zinc metalloendopeptidases.

Abstract

An unusual active site has been identified in a family of zinc metalloendopeptidases that includes bacterial protease III and the human and Drosophila insulin-degrading enzymes. All of these enzymes have been characterized as metalloendopeptidases and purified protease III has been shown to contain stoichiometric levels of zinc. However, all three proteases lack the consensus sequence (HEXXH) described in the active site of other zinc metalloendopeptidases. Instead, these proteases contain an inversion of this motif, HXXEH. To determine whether this region could represent the active site in these proteins, the two histidines in protease III were individually mutated to arginine and the glutamate was mutated to glutamine. All three mutants were devoid of proteolytic activity toward an exogenous substrate, insulin, as compared to the wild-type protease. Three lines of evidence indicate that this loss of activity in the mutants is not due to distortion of the three-dimensional structure of the protein: (i) the mutants are secreted into the periplasmic space and chromatograph normally; (ii) all three mutants are expressed at levels nearly identical to wild-type protein and do not appear to have an increased susceptibility to proteolysis in the bacteria; and (iii) the mutants compete equally with wild-type protein in a radioimmunoassay. The purified wild-type and glutamate mutants were found to contain stoichiometric amounts of zinc by atomic absorption spectrophotometry, whereas both histidine mutants had negligible zinc signals. These findings are consistent with this region being the active site in this protein, with the histidine residues coordinating the essential zinc atom and the glutamate involved in catalysis.