INVERSION OF LIPASE STEREOSPECIFICITY FOR FLUOROGENIC ALKYLDIACYL GLYCEROLS - EFFECT OF SUBSTRATE SOLUBILIZATION

We synthesized enantiomeric 1-O-alkyl-2,3-diacyl-sn-glycerol and 3-O-a lkyl-1,2-diacyl-sn-glycerol containing pyrene as a fluorescent reporte r and the trinitrophenylamino residue as a fluorescence quencher; both reporter groups were covalently bound to the omega end of the acyl ch ains at positions sn-2 and sn-3(1), respectively. The fluorescence of the intact substrate molecules was very low, Chemical or enzymic relea se of the fatty acyl chains lead to fluorescence dequenching. The rate of lipolysis could be measured from the time-dependent increase in fl uorescence intensity. We used the respective substrates for the contin uous determination of activity and stereopreference of four different microbial lipases from Chromobacterium viscosum, Candida rugosa, Pseud omonas sp., Rhizopus arrhizus, as well as cutinase from Fusarium solan i 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 h ydrolysed the sn-l acyl ester bond, if the lipid analog was dispersed in albumin-containing Tris/HCl buffer in the absence of detergent or o rganic solvent. In mixtures of 1:1 (by vol.) water/ethanol, the enzyme s showed higher activity toward the sn-3 acyl ester bond, except for l ipoprotein lipase which preferred the sn-1 acyl isomer under all condi tions tested. Different stereopreferences were observed with the diffe rent lipases if the substrate was solubilized by amphiphiles (micelles of odecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). C. rugosa lipas e and F. solani cutinase showed high stereopreference for the sn-3 acy l ester, whereas Pseudomonas sp. lipase and C. viscosum lipase hydroly sed both enantiomers at similar rates. From spectroscopic studies, it can be inferred that the conformation of the fluorescent lipids is pro bably similar in water, mixtures of water and organic solvents, and in micelles. The possible effect; of reaction conditions on substrate ac cessibility and enzyme conformation on stereoselectivity of the respec tive lipases are discussed.