G. Zandonella et al., Interactions of fluorescent triacylglycerol analogs covalently bound to the active site of a lipase from Rhizopus oryzae, EUR J BIOCH, 262(1), 1999, pp. 63-69
Fluorescent triacylglycerol analogs were synthesized as covalent inhibitors
of lipase activity. The respective 1(3),2-O-dialkylglycero-3(1)-alkyl-phos
phonic acid p-nitrophenyl esters contain a fluorescent pyrenealkyl chain an
d a long-chain alkyl residue bound to the sn-2 and sn-1(3) positions of gly
cerol, respectively. The phosphonic acid p-nitrophenyl ester bond is suscep
tible to nucleophilic substitution by the active serine residue in the cata
lytic triad of a lipase, leading to inactivation of the enzyme. The fluores
cent 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 sep
aration and the (3-O-hexadecyl-2-O-pyrenedecylsn-glycero)-O-(p-nitrophenyl)
-n-hexyl-phol second peak during HPLC separation were found to be potent li
pase inhibitors. After incubation of an equimolar amount of these isomers w
ith 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 fluorescenc
e resonance energy transfer. The extent of tryptophan fluorescence quenchin
g and the concomitant increase in pyrene fluorescence upon excitation of li
pase tryptophans was found to be similar for the above-mentioned isomers. T
hus, the (labeled) sn-2 alkyl chains of a triacylglycerol analog are likely
to interact with the same binding site of the R. oryzae lipase, irrespecti
ve of their steric configuration. However, it was shown that the extent of
resonance energy transfer is strongly influenced by the reaction medium, in
dicating conformational changes of the lipase in different environments.