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  • Molecular Basis for Enantioselectivity of Lipase fromPseudomonas cepacia toward Primary Alcohols. Modeling,Kinetics, and Chemical Modification of Tyr29 to Increase orDecrease Enantioselectivity
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  • Lipase from Pseudomonas cepacia (PCL) shows good enantioselectivity toward primary alcohols.An empirical rule can predict which enantiomer of a primary alcohol reacts faster, but there is noreliable strategy to increase the enantioselectivity. We used a combination of molecular modelingof lipase−transition state analogue complexes and kinetic measurements to identify the molecularbasis of the enantioselectivity toward two primary alcohols: 2-methyl-3-phenyl-1-propanol, 1, and2-phenoxy-1-propanol, 2. In hydrolysis of the acetate esters, PCL favors the (S)-enantiomer of bothsubstrates (E = 16 and 17, respectively), but, due to changes in priorities of the substituents, the(S)-enantiomers of 1 and 2 have opposite shapes. Computer modeling of transition state analoguesbound to PCL show that primary alcohols bind to PCL differently than secondary alcohols. Modelingand kinetics suggest that the enantioselectivity of PCL toward 1 comes from the binding of themethyl group at the stereocenter within a hydrophobic pocket for the fast-reacting enantiomer,but not for the slow-reacting enantiomer. On the other hand, the enantioselectivity toward 2 comesfrom an extra hydrogen bond between the phenoxy oxygen of the substrate to the phenolic OH ofTyr29. This hydrogen bond may slow release of the (R)-alcohol and thus account for the reversalof enantioselectvity. To decrease the enantioselectivity of PCL toward 2-acetate by a factor of 2 toE = 8, we eliminated the hydrogen bond by acetylation of the tyrosyl residues with N-acetylimidazole. To increase the enantioselectivity of PCL toward 2-acetate by a factor of 2 to E =36, we increased the strength of the hydrogen bond by nitration of the tyrosyl residues withtetranitromethane. This is one of the first examples of a rationally designed modification of a lipaseto increase enantioselectivity.
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