Abstract
Endopeptidase EC 3.4.24.15 (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 such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction. In studying the structure and function of EP24.15, we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis. Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray 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 the importance, for binding and catalysis, of the residues (His473, Glu474, and His477) within this motif. A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His473 and His477, has been identified as Glu502. Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes.
Highlights
Endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads
Sequence Alignment with Bacterial Homologues—Computeraided homology searching between functional domains of EP24.15 with those of thermolysin, bacterial elastase, and neutral protease, bacterial homologues previously solved by x-ray crystallography, reveals conserved structural and catalytic elements
These include an HEXXH motif conserved within an active site ␣-helix (His473, Glu474, and His477 in EP24.15, equivalent to His142, Glu143, and His146 in thermolysin), and a glutamate residue, 20 amino acids carboxyl to this motif (Glu497 in EP24.15, equivalent to Glu166 in thermolysin) (Fig. 1)
Summary
The coordination sphere with a long polypeptide loop that further aligns protein residues with the active site zinc. Sequence comparison reveals that a number of key amino acid residues known to be involved in this catalytic mechanism in the aforementioned bacterial enzymes are conserved within a similar sequential context within the carboxyl half of EP24.15 and other mammalian homologues (Fig. 1) These include an HEXXH motif (the histidines representing the first and second ligands, respectively, and the glutamate providing the activated water molecule) in addition to a glutamate residue 20 amino acids carboxyl to this motif (possibly representing the third zinc ligand).. The competitive EP24.15-specific active site-directed inhibitor, cFP-AAY-pAB [12], was employed as a probe to assess the three-dimensional integrity of the catalytic site in both wild type and mutant enzymes These studies have been conducted in parallel with the ongoing structural determination of EP24.15 and are crucial in assessing its catalytic mechanism and role with respect to bioactive neuropeptide substrates.
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