To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with Sc- and Rc-(Rp,Sp)-1,2-dialkylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by Rc-(Rp,Sp)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (Rc-trioctyl) with an inactivation half-time of 75 min, while that for the Sc-(Rp,Sp)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (Sc-trioctyl) compound was 530 min. X-ray structures were obtained of P. cepacia lipase after reaction with Rc-trioctyl to 0.29-nm resolution at pH 4 and covalently modified with Rc-(Rp,Sp)-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butyl-phosphonate (Rc-tributyl) to 0.175-nm resolution at pH 8.5. The three-dimensional structures reveal that both triacylglycerol analogues had reacted with the active-site Ser87, forming a covalent complex. The bound phosphorus atom shows the same chirality (Sp) in both complexes despite the use of a racemic (Rp,Sp) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of Rc-tributyl-complexed P. cepacia lipase, the diacylglycerol moiety has been lost due to an aging reaction, and only the butyl phosphonate remains visible in the electron density. In the Rc-trioctyl complex the complete inhibitor is clearly defined; it adopts a bent tuning fork conformation. Unambiguously, four binding pockets for the triacylglycerol could be detected: an oxyanion hole and three pockets which accommodate the sn-1, sn-2, and sn-3 fatty acid chains. Van der Waals' interactions are the main forces that keep the radyl groups of the triacylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn-2 chain contributes to fixing the position of the inhibitor.
1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversible inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a concomitant release of one mole of p-nitrophenol per mole of enzyme. With both lipases a higher reactivity was observed when the alkyl substituent on the phosphonate is a methyl rather than an octyl chain. Both lipases are highly selective for the chirality of these compounds at glycerol and at phosphorus. Rapid inactivation at an inhibitor concentration of 0.1 mol% in 100 mM NaTDOC (t 1/2 < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S) configuration react 4-10-fold more slowly. The isomer with the p-nitrophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t 1/2 > 1 day) the lipases. These results reflect the known positional- and stereopreference of these enzymes which preferentially release the fatty acid at sn-3 of natural triacylglycerols. The enzymes appeared to be even more selective for the chirality at phosphorus, the differences in reactivity of the faster and slower reacting isomers being as high as about 250-fold for the methylphosphonates and about 60-fold for the octylphosphonates. These phosphonates can be regarded as true active site-directed inhibitors. The inhibited enzymes can be considered as analogues of the tetrahedral intermediate in the acylation step that occurs during triacylglycerol hydrolysis.
