[en] carboxypeptidases/*metabolism ; chemical phenomena ; chemistry ; endopeptidases ; oligopeptides/*metabolism ; probability ; protein conformation ; serine-type d-ala-d-ala carboxypeptidase ; structure-activity relationship ; substrate specificity
[en] The tripeptide Nα,Nɛ-diacetyl-l-lysyl-d-alanyl-d-alanine (Ac2- l-LLys1-dAIa2-dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d-alanyl-d-alanine peptidases, and several ldd tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short-range and long-range interactions. Among the many backbone conformers of minimal energy of the øii space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90% or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ∑P values are still as high as 35–50%. All the above tripeptides bind to the serine d-alanyl-d-alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+d-alanyl-d-alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1, 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2-lLys-dAla-dAla show that the main backbone has a tendency to adopt a ring-like shape from which the lLYS side chain protrudes as an extended structure. This latter structure forms with the C-terminal d-alanyl-d-alanine an angle varying between 120° and 180° (depending on the conformers) so that its N-terminal acetyl group is about 1–1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a dAla at position 2 with both d-alanyl-d-alanine peptidases and at position 3 in the case of the serine d-alanyl-d-alanine peptidase. Finally, all the conformers of the lAla2 and lAla3 analogues of Ac2-lLys-dAla-dAla fall outside the backbone conformational space that comprises the φiφi angles exhibited by the four types of conformers of the ldd tripeptides. The lAla2 and lAla3 tripeptide analogues do not bind to the serine d-alanyl-d-alanine peptidase (at least at a 10 mM concentration) but they behave as noncompetitive inhibitors of the Zn2+d-alanyl-d-alanine peptidase.
Fonds de la Recherche Scientifique Médicale - FRSM ; Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS ; National Institutes of Health - NIH