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 ‐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.