Conformational Analysis of Peptide Substrates and Inhibitors of the Zn²⁺ G and Serine R61 d‐Alanyl‐d‐alanine Peptidases

Abstract

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.

Conformation–activity relationships and the mechanism of action of penicillin

“…Thus, C6 of penicillin, which has the L configuration, must correspond to the asymmetric centre of Ala' , which has the D configuration. However, 6-epipenicillins (8) are inactive (9), and Ac2-L-Lys-L-Ala-D-Ala is not a substrate for PBPs (10). Since the orientation and nature of the acylamino side chain are critical to the activity of penicillin, a structural analogy between penicillin and 2 based on Model A therefore requires that the la' methyl group correspond to H6 of penicillin in the conformations that bind to the active site of PBP.…”

Calculations on the low energy conformers of N-acetyl-D-alanyl-D-alanine

“…Subsequently, the former structures were minimized, Figure 2 shows the four 6-31 / G* from Streptomyces R61, and to the global minimum When comparing the different structures obtained by the four methodologies, the following obtained by De Coen et al 9 with values for the c 3 , f 3 , c 2 , and f 2 dihedrals of 20Њ, 160Њ, 0160Њ, and should be taken into account: the obtained global minimum, energy order of the local minima, and 160Њ, respectively.…”

“…Nevertheless, comconformations of the NAG-NAM disaccharide and the pentapeptide, using the Momany and the Scherparative results regarding the former minimum energy conformations and that of the b-lactam antibiaga force field. 8 De Coen et al, 9 by a force field determined by themselves, performed a conformaotics show that the selected peptide is perfectly adequate for this kind of study. tion analysis of the Ac 2 -L-Lys-D-Ala-D-Ala tripeptide; however, only the most stable conformer was Finally, it should be outlined that the use of three different theoretical methodologies in order reported.…”

Conformational analysis of bacterial cell wall peptides indicates how particular conformations have influenced the evolution of penicillin‐binding proteins, β‐lactam antibiotics and antibiotic resistance mechanisms

“…1); (De Coen et al, 1981;Lamotte et al, 1991). Ac-L-Lys(Ac)-D-Ala-D-Ala and Ac-L-Lys(Ac)-D-Ala-D-Lac were used here to model the conformational profiles displayed by cell wall peptides of wild-type and vancomycin-resistant bacteria, respectively.…”

“…The action of b-lactam antibiotics has been attributed to their structural similarity to natural peptide substrates of transpeptidases, which allows them to be recognized by these enzymes and to inhibit them through formation of a covalent acyl-enzyme complex (Virudachalam and Rao, 1977;Frau et al, 1998;Frau and Price, 1996;Labischinski et al, 1985;Wolfe et al, 1988). In early work, Ac-L-Lys(Ac)-D-Ala-D-Ala was established as a model substrate for bacterial transpeptidase, a feature sometimes overlooked in later studies (De Coen et al, 1981;Frau and Price, 1996).…”