SUBSTITUTION OF POTENTIAL METAL-COORDINATING AMINO-ACID-RESIDUES IN THE ZINC-BINDING SITE OF ENDOPEPTIDASE-24.11

Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane-bound zinc-met allopeptidase. The catalytic zinc ion is coordinated to three amino ac id residues (His538, His587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal-coordinating amino ac id residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure . NEP mutants at positions 583 and 587 were devoid of catalytic activi ty. However, Glu587 NEP and Cys583 NEP were able to bind partially a t ritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants K(m) values were unaltered but k( cat) values were decreased by about 20-fold. Both mutants bound the tr itiated inhibitor with K(d) values similar to that of the wild-type en zyme. Our data suggest that neither histidine-583 nor -587 can be repl aced by any other ligands. On the other hand, the glutamic acid at pos ition 646 can be converted to an aspartic acid or a cysteine indicatin g the importance of a negative charge at this position.