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 Pl' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the PI to P3 sites were tested as substrates. A wide variety of substitutions in Pl' had little effect on K(M) (174-339-mu-M) but markedly affected k(cat) (51.5 s-1 = A > S > R > F > K > T > E = 0). Substitutions in PI had a greater effect on K(M) (366-mu-M-2.46 mM) and also strongly affected k(cat) (98.5 s-1 = A > F >> L > E > 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. It is proposed that conformational constraints at the X6-Pro7 bond affect bradykinin hydrolysis. A general preference for the naturally occurring bradykinin core sequence was observed, which is consistent with the possibility that bradykinin is a physiological substrate for meprin-A. A total of 15 amino acid hydroxamates were tested for inhibition of meprin-A and had K(i) values ranging from 24-mu-M for tyrosine hydroxamate to 1.8 mM for beta-aspartic acid hydroxamate. The bradykinin product analogue peptides AcRPGY and AcRPGY-NHOH were found to be very good inhibitors of meprin-A hydrolysis with K(i) values of 15.6 and 3.7-mu-M, respectively. Both tetrapeptides inhibited via a simple noncompetitive mechanism, suggesting the possible existence of regulatory binding sites.