Interactions of fluorescent triacylglycerol analogs covalently bound to the active site of a lipase from Rhizopus oryzae

Abstract

Fluorescent triacylglycerol analogs were synthesized as covalent inhibitors of lipase activity. The respective 1(3),2-O-dialkylglycero-3(1)-alkyl-phosphonic acid p-nitrophenyl esters contain a fluorescent pyrenealkyl chain and a long-chain alkyl residue bound to the sn-2 and sn-1(3) positions of glycerol, respectively. The phosphonic acid p-nitrophenyl ester bond is susceptible to nucleophilic substitution by the active serine residue in the catalytic triad of a lipase, leading to inactivation of the enzyme. The fluorescent dialkylglycerophosphonates contain two chiral centers, the sn-2 carbon of glycerol and the phosphorus atom. The (1-O-hexadecyl-2-O-pyrenedecyl-sn-glycero)-O-(p-nitrophenyl)-n-hexyl-phosphonate, first peak during HPLC separation and the (3-O-hexadecyl-2-O-pyrenedecylsn-glycero)-O-(p-nitrophenyl)-n-hexyl-phosphonate, second peak during HPLC separation were found to be potent lipase inhibitors. After incubation of an equimolar amount of these isomers with lipase from Rhizopus oryzae complete inactivation was observed. Stable conjugates containing a 1 : 1 molar ratio of lipid to protein were formed. The spatial proximity of the fluorescently labeled sn-2 alkyl chain of the inhibitor and tryptophan residues of the lipase was assessed by fluorescence resonance energy transfer. The extent of tryptophan fluorescence quenching and the concomitant increase in pyrene fluorescence upon excitation of lipase tryptophans was found to be similar for the above-mentioned isomers. Thus, the (labeled) sn-2 alkyl chains of a triacylglycerol analog are likely to interact with the same binding site of the R. oryzae lipase, irrespective of their steric configuration. However, it was shown that the extent of resonance energy transfer is strongly influenced by the reaction medium, indicating conformational changes of the lipase in different environments.Keywords: fluorogenic triacylglycerol analogs; microbial lipases; phosphonate inhibitors; pyrene and tryptophan fluorescence; resonance energy transfer.In many cases the lipase-catalyzed hydrolysis of triacylglycerols is a stereoselective process, the extent of stereoselectivity depending on the enzyme [1], the substrate [1,2], the solvent [3±7] and the physicochemical properties of these systems. For example, studies on monolayers at the air±water interface have demonstrated that lipase activity and stereoselectivity are influenced in opposite ways, depending on the surface pressure applied [8]. Accordingly, lipase function depends critically on the reaction medium, which influences the structure of the protein and its interaction with the substrate. Knowledge about molecular mechanisms of protein±lipid interaction is essential for an understanding of lipase stereoselectivity. X-ray crystallography studies on lipase±inhibitor complexes provided detailed information about lipase±substrate interaction at a submolecular level [9±13]. However, to date no direct experimental evidence is available about enzyme±substrate interaction in solution.Inhibitors are useful tools fo...

Site‐Selective Ser‐Hydrolase Labelling with a Luminescent Organometallic NCN–Platinum Complex

“…to 40°C) and time (12 h to 5 d). Based on 31 P and 1 H NMR spectroscopic analysis, none of these screens gave rise to any formation of the desired product. This was rather surprising, since other NCN-platinum-type complexes that contained acetylene moieties were successfully applied under standard click reaction conditions in our laboratory before.…”

“…[61,63] The subsequent amide coupling reaction of 5 with 2 in the presence of benzotriazole-1-yloxytris-(dimethylamino)phosphonium hexafluorophosphate (BOP) gave rise to the formation of target molecule 6, which was isolated in 77 % overall yield starting from 4. Complex 6 was characterized by 1 H, 31 P, 13 C NMR and IR spectroscopy, high-resolution mass spectrometry and by photophysical methods (vide infra). Notably, both the organometallic complex 2 and phosphonate 5 did not show any signs of decomposition under the applied reaction conditions.…”

“…[21] Phosphonates have long been known to be substrate analogues for serine hydrolases, and even phosphonates with various organic fluorescent reporter tags have been studied by different groups. [17,22,[29][30][31] So far, phosphonates substituted by organic dyes [e.g., fluorescent dansyl (Figure 1), [22] rhodamine [15] and p-nitrobenzofurazan [32] groups] have [20,21] of an organic luminescent dansyl phosphonate inhibitor (middle) [22] and of the organometallic luminescent target inhibitor 6 (right).…”

Σύνθεση Και Μελέτη Νέων Βιοδραστικών Λιπιδίων

“…17 Σχήμα 2.7. Πορεία παρασκευής φωσφονικών εστέρων που αναστέλλουν την κουτινάση και SHL λιπάση.Η Φωσφονικά παράγωγα με δομή ανάλογη της τριακυλογλυκερόλης, όπου ο εστερικός δεσμός στην sn-1 ή sn-3 θέση έχει αντικατασταθεί με φωσφονικό εστέρα της π-νιτρο-φαινόλης (σχήμα 2.8), εμφανίζουν ανασταλτική δράση για τις λιπάσες από Chromobacterium viscosum (CVL)62 και Rhizopus oryzae (ROL) 62,63,64.…”