Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Abstract

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.