Mapping the active site of meprin-A with peptide substrates and inhibitors.

The extended substrate-binding site of meprin-A, a tetrameric metalloendopeptidase from brush border membranes of mouse kidney proximal tubules, was mapped with a series of peptide substrates. Previous studies led to the development of the chromogenic substrate Phe5(4-nitro)bradykinin for meprin-A. With this substrate, several biologically active peptides were screened as alternate substrate inhibitors, and, of these, bradykinin (RPPGFSPFR) was found to be the best substrate with a single cleavage site (Phe5-Ser6). Three types of bradykinin analogues were used for a systematic investigation of substrate specificity: (1) nonchromogenic bradykinin analogues with substitutions in the P3 to P3' subsites were used as alternative substrate inhibitors of nitrobradykinin hydrolysis, (2) analogues of nitrobradykinin with variations in the P1' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the P1 to P3 sites were tested as substrates. A wide variety of substitutions in P1' had little effect on KM (174-339 microM) but markedly affected kcat (51.5 s-1 = A greater than S greater than R greater than F greater than K greater than T greater than E = 0). Substitutions in P1 had a greater effect on KM (366 microM-2.46 mM) and also strongly affected kcat (98.5 s-1 = A greater than F much greater than L greater than E greater than K = 2.4 s-1). The variety of allowed cleavages indicates that meprin-A does not have strict requirements for residues adjacent to the cleavage site. Substitutions farther from the scissle bond also affected binding and hydrolysis, demonstrating that multiple subsite interactions are involved in meprin-A action.(ABSTRACT TRUNCATED AT 250 WORDS)