This paper reports the chiroptical properties of thionated N‐acyl amino acid and N‐acyl dipeptide N′‐methylamide models. It was found that the optical activity of the thioamide chromophore is dominated by the chiral contribution of perturbants attached to Cα at the N‐H side of the thioamide group. The appearance of a strong negative ππ* band near 270 nm is indicative of the semiextended conformation of this residue. The ϕ ∼ −70°, ψ ⩾ 120° set of torsion angles is compatible with a type II βt‐turn or a γt‐turn conformation with the perturbing N‐H side residue in the i + 1 position of the turn. (The subscript t or tt denotes that one or both of the H‐bonded moieties is thioamide.) Earlier data show that both βt‐ and γt‐turns may be fixed by CS ⃛H‐N(CO) intramolecular H bonds. The appearance of one or two weak nπ* bands and a positive ππ* band at about 270 nm is characteristic of type II βt‐turns containing the H‐bonded thioamide group attached to the glycine residue in position i + 2. The extended conformation (ϕ ∼ −140°, ψ ∼ 140°) of a residue after the thioamide group gives rise to a negative nπ* and a positive ππ* band of comparable magnitude. Peptid1e sequences with alternating thioamide–amide–thioamide backbone tend to adopt 1t ⇆ 4t H‐bonded βtt conformations. CD studies show that type II βtt‐turns have unique chiroptical properties: the ππ* region is dominated by an exceedingly strong negative band near 260 nm (|Δε| = 19–24) accompanied by a weaker band at higher wavelength values.
The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(beta-aspartyl)-2-acetamido-2-deoxy-beta-D-glucopyranosyla min e. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A "CD additivity" experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH3 and 1-N-acetyl-3,4,6- tri-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the "spectroscopic sum") was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety ("mathematical sum"). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I beta-turn content. Simultaneously, the type II beta-turn conformational percentage increased to approximately 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation.
Prolyl oligopeptidase, a member of the new family of serine proteases, exhibits significant mechanistic differences compared with the enzymes of the chymotrypsin and subtilisin families. Our kinetic study using the thiono substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2naphthylamide suggests that the putative oxyanion binding site is important in prolyl oligopeptidase catalysis, although to a lesser extent than in the chymotrypsinand subtilisin-catalyzed reactions. By using another thiono substrate, benzyloxycarbonyl-Gly[CS-NH]Pro-2-naphthylamide, it is demonstrated that the distant S2P2 hydrogen bond (formed between the S2 subsite and P2 peptide residue) makes a greater contribution to catalysis than does stabilization by the oxyanion binding site involved directly in the bond cleavage. In contrast to the reactions catalyzed by chymotrypsin and subtilisin, no kinetic deuterium isotope effect is apparent in the acylation of prolyl oligopeptidase measured either with the specific benzyloxycarbonyl-Gly-Pro-2naphthylamide, or with the very poor substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide. This indicates that the rate-limiting conformational change is induced by the substrate.
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