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.
Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses
triglycerides efficiently All the inhibited forms of lipolytic enzymes described
so far are based on the use of small organophosphate
and organophosphonate inhibitors which bear little resemblance to a natural
triglyceride substrate In this article we describe the crystal structure
of cutinase covalently inhibited by R)-1,2-dibutyl carbamoylglycero-3-O-p-nitrophenylbutyl-phos phonate a triglyceride
analogue mimicking the first tetrahedral intermediate along the reaction pathway
The structure which has been solved at 2.3 A reveals
that in both the protein molecules of the asymmetric unit
the inhibitor is almost completely embedded in the active site
crevice The overall shape of the inhibitor is that of
a fork the two dibutyl carbamoyl chains point towards the
surface of the protein whereas the butyl chain bound to
the phosphorous atom is roughly perpendicular to the sn-1 and
sn-2 chains The sn-3 chain is accommodated in a rather
small pocket at the bottom of the active site crevice
thus providing a structural explanation for the preference of cutinase
for short acyl chain substrates