Peter Carlqvist

Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions

Michael‐type additions of various thiols and α,β‐unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild‐type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active‐site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael‐type additions was initially explored by quantum‐chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular‐dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The kcat values were found to be in the range of 10−3 to 4 min−1, similar to the values obtained with aldolase antibodies. The enzyme proficiency was 107. Furthermore, the Michael‐type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single‐point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of α,β‐unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step‐mechanism with formation of an oxyanionic intermediate. The active‐site residue His224 functions as a general acid‐base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

Local Electron Attachment Energy and Its Use for Predicting Nucleophilic Reactions and Halogen Bonding

A new local property, the local electron attachment energy [E(r)], is introduced and is demonstrated to be a useful guide to predict intermolecular interactions and chemical reactivity. The E(r) is analogous to the average local ionization energy but indicates susceptibility toward interactions with nucleophiles rather than electrophiles. The functional form E(r) is motivated based on Janaks theorem and the piecewise linear energy dependence of electron addition to atomic and molecular systems. Within the generalized Kohn-Sham method (GKS-DFT), only the virtual orbitals with negative eigenvalues contribute to E(r). In the present study, E(r) has been computed from orbitals obtained from GKS-DFT computations with a hybrid exchange-correlation functional. It is shown that E(r) computed on a molecular isodensity surface, ES(r), reflects the regioselectivity and relative reactivity for nucleophilic aromatic substitution, nucleophilic addition to activated double bonds, and formation of halogen bonds. Good to excellent correlations between experimental or theoretical measures of interaction strengths and minima in ES(r) (ES,min) are demonstrated.