Zandonella_1995_Eur.J.Biochem_231_50

We synthesized enantiomeric 1-O-alkyl-2,3-diacyl-sn-glycerol and 3-O-alkyl-1,2-diacyl-sn-glycerol containing pyrene as a fluorescent reporter and the trinitrophenylamino residue as a fluorescence quencher; both reporter groups were covalently bound to the omega end of the acyl chains at positions sn-2 and sn-3(1), respectively. The fluorescence of the intact substrate molecules was very low. Chemical or enzymic release of the fatty acyl chains lead to fluorescence dequenching. The rate of lipolysis could be measured from the time-dependent increase in fluorescence intensity. We used the respective substrates for the continuous determination of activity and stereopreference of four different microbial lipases from Chromobacterium viscosum, Candida rugosa, Pseudomonas sp., Rhizopus arrhizus, as well as cutinase from Fusarium solani and lipoprotein lipase from bovine milk. The stereopreference of the lipases depended, in general, on how the substrate was solubilized in the reaction medium. All lipases under investigation preferentially hydrolysed the sn-1 acyl ester bond, if the lipid analog was dispersed in albumin-containing Tris/HCl buffer in the absence of detergent or organic solvent. In mixtures of 1:1 (by vol.) water/ethanol, the enzymes showed higher activity toward the sn-3 acyl ester bond, except for lipoprotein lipase which preferred the sn-1 acyl isomer under all conditions tested. Different stereopreferences were observed with the different lipases if the substrate was solubilized by amphiphiles (micelles of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). C. rugosa lipase and F. solani cutinase showed high stereopreference for the sn-3 acyl ester, whereas Pseudomonas sp. lipase and C. viscosum lipase hydrolysed both enantiomers at similar rates. From spectroscopic studies, it can be inferred that the conformation of the fluorescent lipids is probably similar in water, mixtures of water and organic solvents, and in micelles. The possible effects of reaction conditions on substrate accessibility and enzyme conformation on stereoselectivity of the respective lipases are discussed.