Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with S-c-and R-c-(R-p,S-p)-1,2-dialkylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by R-c-(R-p,S-p)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (R-c-trioctyl) with an inactivation half-time of 75 min, while that for the S-c-(R-p,S-p)-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (S-c-trioctyl) compound was 530 min. X-ray structures were obtained of P. cepacia lipase after reaction with R-c-trioctyl to 0.29-nm resolution at pH 4 and covalently modified with R-c-(R-p,S-p)-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butyl-phosphonate (R-c-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 (S-p) in both complexes despite the use of a racemic (R-p,S-p) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of R-c-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 R-c-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-l, 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.