title: Structural basis of the chiral selectivity of Pseudomonas cepacia lipase.
abstract: 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.
authors.affiliation: BIOSON Research Institute and Laboratory of Biophysical Chemistry, University of Groningen, The Netherlands.
authors.forename: D A
authors.initials: DA
authors.lastname: Lang
authors.forename: M L
authors.initials: ML
authors.lastname: Mannesse
authors.forename: G H
authors.initials: GH
authors.lastname: de Haas
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
authors.forename: B W
authors.initials: BW
authors.lastname: Dijkstra
mesh_terms: D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008024:Ligands; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013237:Stereoisomerism; D014280:Triglycerides / Q000737:chemistry
chemical_list: D008024:Ligands; D014280:Triglycerides; D008049:Lipase