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Substitution of potential metal‐coordinating amino acid residues in the zinc‐binding site of endopeptidase‐24.11
Author(s) -
Le Moual Hervé,
Roques Bernard P.,
Crine Philippe,
Boileau Guy
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81392-d
Subject(s) - chemistry , aspartic acid , zinc , cysteine , histidine , endopeptidase , amino acid , glutamic acid , stereochemistry , mutant , enzyme , biochemistry , organic chemistry , gene
Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane‐bound zinc‐metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His 538 , Hi 587 and Glu 646 ) and a water molecule. Here, we have systematically substituted potential metal‐coordinating amino acid 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 activity. However, Glu 587 NEP and Cys 583 NEP were able to bind partially a tritiated 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 tritiated inhibitor with K d values similar to that of the wild‐type enzyme. Our data suggest that neither histidine‐583 nor ‐587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.