Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15 - Identification of active site histidine and glutamate residues

Endopeptidase EC 5.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads. Its substrate specificity includes a number of physiologically important neuropeptides s uch as neurotensin, bradykinin, and gonadotropin-releasing hormone, the pri ncipal regulatory peptide for reproduction. In studying the structure and f unction of EP24.15, we have employed in. vitro mutagenesis and subsequent p rotein expression to genetically dissect the enzyme and allow us to glean i nsight into the mechanism of substrate binding and catalysis, Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-r ay crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues. The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes, Mutations have confirmed th e importance, for binding and catalysis, of the residues (His(473), Glu(474 ), and His(477)) within this motif, A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His(473 ) and His(477), has been identified as Glu(502). Conservative alterations t o these residues drastically reduces enzymatic activity against both a puta tive physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(R S) -carboxy-3-phenylpropyl] -Ala-Ala-Tyr-p-aminobenzoate, the binding of wh ich we have shown to be dependent upon the presence, and possibly coordinat ion, of the active site zinc ion. These studies contribute to a more comple te understanding of the catalytic mechanism of EP24.15 and will aid in rati onal design of inhibitors and pharmacological agents for this class of enzy mes.