Glycyl-histidyl-lysine (GHK) is a quencher of alpha,beta-4-hydroxy-trans-2-nonenal: a comparison with carnosine. insights into the mechanism of reaction by electrospray ionization mass spectrometry, 1H NMR, and computational techniques.
Histidine-containing oligopeptides are currently studied as detoxifying agents against cytotoxic alpha,beta-unsaturated aldehydes (prototype: 4-hydroxy-2-nonenal, HNE), electrophilic end products formed by decomposition of omega-6 polyunsaturated fatty acids, associated with severe pathologies such as diabetes, nephropathy, retinopathy, and neurodegenerative diseases. This study evaluated the quenching reaction against HNE of the endogenous tripeptide l-glycyl- l-histidyl- l-lysine (GHK), an oligopeptide discovered to be a growth-modulating factor and also a strong activator of wound healing. We first evaluated the HNE consumption (50 microM, HPLC-UVDAD method) in the presence of GHK (1 mM) in physiomimetic conditions (phosphate buffer, pH 7.4) and confirmed GHK/HNE adduct formation by mass spectrometric analysis (ESI-MS/MS) and (1)H NMR analyses. These results indicated that GHK was an effective quencher of HNE, although significantly less potent than the reference compound carnosine, and that HNE modulation by GHK can contribute to the satisfactory outcome of the wound-healing process. In the second part of the study, we investigated the quenching reaction between GHK and HNE, in parallel to carnosine, using (1)H NMR and computational analyses. At a mechanistic level, this explained the different reactivity of the two peptides: (i) The greater stability of the macrocyclic intermediate HNE/carnosine was compared to HNE/GHK. (ii) GHK in solution has a quasi-folded conformation due to the interaction of four intramolecular hydrogen bonds, three of which need to be broken for the transition state to form (energy barrier, approximately 20 kcal/mol). By contrast, carnosine, with an extended conformation and only one hydrogen bond, requires less energy to reach the transition state ( approximately 7 kcal/mol). (iii) The different stereoelectronic features of the transition state lead to the intramolecular Michael reaction, that is, the favorable superimposition of carnosine highest occupied molecular orbital and the HNE lowest unoccupied molecular orbital, in relation to the unfavorable orbital configuration of GHK. The overall findings provide interesting and useful insights into the mechanisms of interaction of both GHK and carnosine with HNE and illustrate the utility of computational studies for defining the (optimal) chemical and structural parameters for an optimal quenching of alpha,beta-unsaturated aldehydes.
Faculty of Pharmacy, Istituto di Chimica Farmaceutica e Tossicologica Pietro Pratesi, University of Milan, Viale Abruzzi 42, 20131 Milan, Italy. giangiacomo.beretta@unimi.it
Giangiacomo
G
Beretta
Roberto
R
Artali
Luca
L
Regazzoni
Monica
M
Panigati
Roberto Maffei
RM
Facino
D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism; D002336:Carnosine / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D000447:Aldehydes; D009842:Oligopeptides; C013166:2-nonenal; C020905:glycyl-histidyl-lysine; D002336:Carnosine