Charge polarity reversal inverses the specificity of neutral endopeptidase-24.11.
Attempts to change enzyme specificity by charge polarity reversal have so far met with little success, probably due to a destabilization of the resulting ion pair in an environment naturally optimized for the inverted pair. In the zinc metallopeptidase neutral endopeptidase-24.11 (EC 3.4.24.11), Arg102, involved in substrate binding, is probably located at the edge of the active site (Bateman, R.C., Jr., Kim, Y.-A., Slaughter, C., and Hersh, L.B. (1990) J. Biol. Chem. 265, 8365-8368; Beaumont, A., Le Moual, H., Boileau, G., Crine, P., and Roques, B.P. (1991) J. Biol. Chem. 266, 214-220). This environment may be favorable for polarity reversal, as in water the energies of reverse ion pairs would be identical. We show here that, while mutating Arg102 to Glu reduces the specificity of a C-terminally negatively charged substrate 16-fold, it increases that of a substrate with an optimally positioned positive charge 29-fold. The concept of charge polarity reversal can be extended to other zinc metallopeptidases, and the mutated enzyme could also have applications in the enantiomeric separation of unnatural amino acids.
Beaumont
A
A
Département de Chimie Organique (U 266 Institut National de la Santé et de la Recherche Médicale, UA 498 Centre National de la Recherche Scientifique, Paris, France.
Barbe
B
B
Le Moual
H
H
Boileau
G
G
Crine
P
P
Fournié-Zaluski
M C
MC
Roques
B P
BP
D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004563:Electrochemistry; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015244:Thiorphan / Q000031:analogs & derivatives*
D013438:Sulfhydryl Compounds; D016308:Enkephalin, Leucine-2-Alanine; C072559:retrothiorphan; D004247:DNA; D015244:Thiorphan; D015260:Neprilysin