STRUCTURAL BASIS OF THE CHIRAL SELECTIVITY OF PSEUDOMONAS-CEPACIA LIPASE

To investigate the enantioselectivity of Pseudomonas cepacia lipase, i nhibition studies were performed with S-c-and S-p)-1,2-dialkylcarbamoy lglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by S-p)-1,2-di octylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (R-c-trioctyl ) with an inactivation half-time of 75 min, while that for the S-p)-1, 2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (S-c-tri octyl) compound was 530 min. X-ray structures were obtained of P. cepa cia lipase after reaction with R-c-trioctyl to 0.29-nm resolution at p H 4 and covalently modified with 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 triacylgl ycerol analogues had reacted with the active-site Ser87, forming a cov alent 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 moiet y has been lost due to an aging reaction, and only the butyl phosphona te remains visible in the electron density. In the R-c-trioctyl comple x the complete inhibitor is clearly defined; it adopts a bent tuning f ork conformation. Unambiguously, four binding pockets for the triacylg lycerol could be detected: an oxyanion hole and three pockets which ac commodate the sn-l, sn-2, and sn-3 fatty acid chains. Van der Waals' i nteractions are the main forces that keep the radyl groups of the tria cylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn-2 chain contributes to fixing the positi on of the inhibitor.