Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Zandonella, G; Stadler, P; Haalck, L; Spener, F; Paltauf, F; Hermetter, A.
Interactions of fluorescent triacylglycerol analogs covalently bound to the active site of a lipase from Rhizopus oryzae.
Eur J Biochem. 1999; 262(1):63-69
Doi: 10.1046/j.1432-1327.1999.00325.x
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- Co-Autor*innen der Med Uni Graz
- Spener Friedrich
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- 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-hex yl- phosphonate, first peak during HPLC separation and the (3-O-hexadecyl-2-O-pyrenedecyl-sn-glycero)-O-(p-nitrophenyl)-n-hex yl- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Binding Sites -
- Fluorescent Dyes - metabolism
- Hydrolysis -
- Lipase - metabolism
- Rhizopus - enzymology
- Trialkyltin Compounds - metabolism
- Triglycerides - chemistry Triglycerides - metabolism
- Find related publications in this database (Keywords)
- fluorogenic triacylglycerol analogs
- microbial lipases
- phosphonate inhibitors
- pyrene and tryptophan fluorescence
- resonance energy transfer