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-metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, His587 and Glu646) 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, Glu587 NEP and Cys583 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 Km values were unaltered but kcat values were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kd 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.
Le Moual
H
H
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
Roques
B P
BP
Crine
P
P
Boileau
G
G
D001665:Binding Sites; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
D005971:Glutamates; D006898:Hydroxylamines; D008667:Metalloproteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; C046556:N-(3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine; D015260:Neprilysin; D015032:Zinc; D005998:Glycine